Non-oligomerizing tandem fluorescent proteins

ABSTRACT

Non-oligomerizing fluorescent proteins, which are formed by operatively linking two or more monomers of a fluorescent protein, or which are derived from a fluorescent protein having at least one mutation that reduces or eliminates the ability of the fluorescent protein to oligomerize, are provided. The non-oligomerizing fluorescent proteins can be derived from a naturally occurring green fluorescent protein, a red fluorescent protein, or other fluorescent protein, or a fluorescent protein related thereto. Also provided is a fusion protein, which includes a non-oligomerizing fluorescent protein linked to at least one polypeptide of interest. In addition, a polynucleotide encoding a non-oligomerizing fluorescent protein is provided, as is a recombinant nucleic acid molecule, which includes polynucleotide encoding a non-oligomerizing fluorescent protein operatively linked to at least a second polynucleotide. Vectors and host cells containing such polynucleotides also are provided, as are kits containing one or more non-oligomerizing fluorescent proteins or encoding polynucleotides or constructs derived therefrom. Further provided are methods of making and using the proteins and polynucleotides.

[0001] This application is continuation-in-part application of U.S. Ser.No. 09/794,308, filed Feb. 26, 2001, the entire contents of which isincorporated herein by reference.

[0002] This invention was made in part with government support underGrant No. NS 27177 by the National Institute of Neurological Disordersand Stroke and Grant No. GM 62114-01 awarded by the National Instituteof General Medical Sciences. The government has certain rights in thisinvention.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] The present invention relates generally to fluorescent proteins,and more specifically to tandem fluorescent protein homodimers, whichhave a reduced propensity to oligomerize as compared to unlinkedfluorescent protein monomers, and to methods of making and using suchnon-oligomerizing tandem fluorescent proteins.

[0005] 2. Background Information

[0006] The identification and isolation of fluorescent proteins invarious organisms, including marine organisms, has provided a valuabletool to molecular biology. The green fluorescent protein (GFP) of thejellyfish Aequorea victoria, for example, has become a commonly usedreporter molecule for examining various cellular process, including theregulation of gene expression, the localization and interactions ofcellular proteins, the pH of intracellular compartments, and theactivities of enzymes.

[0007] The usefulness of Aequorea GFP has led to the identification ofnumerous other fluorescent proteins in an effort to obtain proteinshaving different useful fluorescence characteristics. In addition,spectral variants of Aequorea GFP have been engineered, thus providingproteins that are excited or fluoresce at different wavelengths, fordifferent periods of time, and under different conditions. Theidentification and cloning of a red fluorescent protein, dsRed, fromDiscosoma raised a great deal of interest due to its ability tofluoresce in the red wavelength. The availability of such fluorescentproteins has greatly expanded the studies that the proteins can be usedfor and, consequently, our understanding of cellular structure andfunction.

[0008] Although the availability of a wide variety of naturallyoccurring fluorescent proteins and spectral variants of the proteins hasallowed for substantial advances, limitations to the use of fluorescentproteins remain. In particular, GFP and its spectral variants, as wellas other naturally occurring fluorescent proteins such as dsRed have apropensity to self-associate under physiological conditions, thusforming dimers, tetramers, and the like. As such, it can be difficult insome cases to confirm whether a result is due, for example, to aspecific interaction of two proteins under investigation, or whether aperceived interaction is an artifact caused by the oligomerization offluorescent proteins linked to each of the two proteins underinvestigation.

[0009] Substantial progress has been made in designing mutants of GFPand its spectral variants that have substantially reduced oligomerizingactivity. Progress also has been made in designing dsRed mutants thathave a reduced propensity to form oligomers, and dsRed mutants that formonly dimers have been developed. However, previous efforts to modifydsRed to prevent dimer formation have resulted in the formation ofnon-fluorescent proteins. Thus, a need exists for methods to reduce thepropensity of red fluorescent proteins such as dsRed to self-associate.The present invention satisfies this need and provides additionaladvantages.

SUMMARY OF THE INVENTION

[0010] The present invention relates to a non-oligomerizing tandemfluorescent protein, ;which includes a first monomer of a fluorescentprotein operatively linked to at least a second monomer of thefluorescent protein, wherein the propensity of the tandem fluorescentprotein to oligomerize is reduced or inhibited as compared to a monomerof the fluorescent protein. The fluorescent protein of anon-oligomerizing tandem fluorescent protein can be a green fluorescentprotein (GFP), a red fluorescent protein (RFP), or a fluorescent proteinrelated to a GFP or an RFP.

[0011] In one embodiment, the fluorescent protein is a Discosoma RFP ora fluorescent protein related to a Discosoma RFP, for example, a DsRedprotein having an amino acid sequence as set forth in SEQ ID NO:12, or amutant of SEQ ID NO:12, such as SEQ ID NO:12 having an I125R mutation.In another embodiment, the fluorescent protein is an Aequorea GFP, aRenilla GFP, a Phialidium GFP, or a fluorescent protein related to anAequorea GFP, a Renilla GFP, and a Phialidium GFP, for example, a cyanfluorescent protein (CFP), or a yellow fluorescent protein (YFP), or aspectral variant of the CFP or the YFP, or an enhanced GFP (EGFP; SEQ IDNO: 4), an enhanced CFP (ECFP; SEQ ID NO: 6), an EYFP-V68L/Q69K (SEQ IDNO: 10), or an enhanced YFP (EYFP; SEQ ID NO: 8), each of which isrelated to Aequorea GFP. In still another embodiment, the fluorescentprotein of a non-oligomerizing tandem fluorescent protein comprises amutation of an amino acid residue corresponding to A206, L221, F223, ora combination thereof of SEQ ID NO: 2, for example, a mutationcorresponding to an A206K mutation, an L221K mutation, an F223Rmutation, or an L221K and F223R mutation of SEQ ID NO: 2, or a mutationcorresponding to an A206K mutation, an L221K mutation, an F223Rmutation, or an L221K and F223R mutation of SEQ ID NO: 6 or SEQ ID NO:10.

[0012] The first monomer and at least second monomer of anon-oligomerizing tandem fluorescent protein are operatively linked suchthat an intramolecular oligomer is formed, thus reducing or inhibitingthe propensity of the fluorescent protein to form intermolecularoligomers. The first and at least second monomer can be operativelylinked using any bond or linker that does not disrupt the fluorescentproperty of the fluorescent protein. In one embodiment, the first and atleast second monomer are operatively linked using a peptide linker, forexample, the peptide linker has an amino acid sequence as set forth inSEQ ID NO:26. For example, the fluorescent protein can have an aminoacid sequence as set forth in SEQ ID NO:12 and the peptide linker has anamino acid sequence as set forth in SEQ ID NO:26, or the fluorescentprotein can have an amino acid sequence substantially as set forth inSEQ ID NO:12, including an I125R mutation, and the peptide linker has anamino acid sequence as set forth in SEQ ID NO:26. In another embodiment,the first and at least second monomer are operatively linked using apolynucleotide sequence, a peptidomimetic, or other synthetic linker,for example, a synthetic polymer.

[0013] A non-oligomerizing tandem fluorescent protein can further atleast a third monomer of the fluorescent protein, which is operativelylinked to the first monomer or the second monomer. The linkers foroperatively linking the three or more monomers of a non-oligomerizingtandem fluorescent protein can be the same or different linkers.

[0014] The present invention also relates to a fusion protein, whichincludes a non-oligomerizing tandem fluorescent protein operativelylinked to at least one polypeptide of interest. The non-oligomerizingtandem fluorescent protein can be linked to the polypeptide of interestusing any linker or linkage, including, for example, a peptide bond, apeptide linker, or other linker molecule. The polypeptide of interestcan be any polypeptide, including, for example, a peptide tag such as apolyhistidine peptide; a cellular polypeptide such as an enzyme, aG-protein, a growth factor receptor, or a transcription factor; or areporter polypeptide, which provides a detectable signal or provides ameans for isolating a fusion protein containing the reporterpolypeptide. The polypeptide of interest also can be one of two or moreproteins that associate to form a complex.

[0015] The present invention further relates to a kit, which contains atleast one non-oligomerizing tandem fluorescent protein of the invention,and can contain a plurality of different non-oligomerizing tandemfluorescent proteins. If desired, one or more non-oligomerizing tandemfluorescent proteins of the kit can be a fusion protein.

[0016] The present invention also relates to a polynucleotide encoding anon-oligomerizing tandem fluorescent protein of the invention, as wellas to a recombinant nucleic acid molecule, which includes apolynucleotide of the invention operatively linked to at least a secondpolynucleotide. The at least second polynucleotide can be, for example,a transcription or translation regulatory element, or can encode apolypeptide of interest, or can include a restriction endonucleaserecognition site or a recombinase recognition site. Also provided arevectors, which contain a polynucleotide or recombinant nucleic acidmolecule of the invention, as well as host cell, which contains such apolynucleotide or vector. In addition, a kit containing at least onepolynucleotide or recombinant nucleic acid molecule of the invention isprovided. In one embodiment, the kit contains a plurality of differentpolynucleotides or recombinant nucleic acid molecules or a combinationthereof.

[0017] The present invention further relates to a tandemnon-oligomerizing fluorescent protein, which includes a donor,comprising a first fluorescent protein, an acceptor, comprising a secondfluorescent protein, and a peptide linker moiety operatively linking thedonor and the acceptor. In such a tandem non-oligomerizing fluorescentprotein, the first fluorescent protein and second fluorescent proteinare different, and at least the first fluorescent protein or the secondfluorescent protein is a non-oligomerizing tandem fluorescent protein ofthe invention. In addition, the cyclized amino acids of the donor emitlight characteristic of the donor, and the donor and the acceptorexhibit fluorescence resonance energy transfer when the donor isexcited, and the linker moiety does not substantially emit light toexcite the acceptor.

[0018] In one embodiment, each of the first fluorescent protein and thesecond fluorescent protein is a non-oligomerizing tandem fluorescentprotein in a tandem non-oligomerizing fluorescent protein of theinvention. For example, the non-oligomerizing tandem fluorescent proteincan be a Discosoma RFP or a fluorescent protein related to a DiscosomaRFP, such as a DsRed protein having an amino acid sequence as set forthin SEQ ID NO: 12 or a mutant DsRed protein such as SEQ ID NO:12containing an I125R mutation.

[0019] In another embodiment, the first fluorescent protein is anon-oligomerizing tandem fluorescent protein, and the second fluorescentprotein is a non-oligomerizing fluorescent protein. Thenon-oligomerizing fluorescent protein can contain a mutation of an aminoacid residue corresponding to A206, L221, F223, or a combination thereofof SEQ ID NO:2, for example, a mutation corresponding toS65G/S72A/T203Y/H23 IL in SEQ ID NO:2; a mutation corresponding toS65G/V68L/Q69KIS72A/T203Y/H231L in SEQ ID NO:2; a mutation correspondingto K26R/F64L/S65T/Y66W/N146I/M153T/V163A/N164H/H231L in SEQ ID NO: 2; ora mutation corresponding to H148G in SEQ ID NO: 2.

[0020] The present invention also relates to a method for determiningthe pH of a sample. Such a method can be performed, for example, bycontacting the sample with a first non-oligomerizing tandem fluorescentprotein, wherein the emission intensity of the first non-oligomerizingtandem fluorescent protein changes as pH varies between pH 5 and pH 10,exciting the indicator; and determining the intensity of light emittedby the first non-oligomerizing tandem fluorescent protein at a firstwavelength, wherein the emission intensity of the firstnon-oligomerizing tandem fluorescent protein indicates the pH of thesample. The sample can be any sample, including, for example, abiological tissue such as a cell or a fraction thereof.

[0021] A method of determining the pH of a sample can include contactingthe sample with a non-oligomerizing fluorescent protein, which isdifferent from the first non-oligomerizing tandem fluorescent protein,and wherein the emission intensity of the non-oligomerizing fluorescentprotein changes as pH varies from 5 to 10, and emits at a secondwavelength that is distinct from the first wavelength; exciting thenon-oligomerizing fluorescent protein; determining the intensity oflight emitted by the non-oligomerizing fluorescent protein at the secondwavelength; and comparing the fluorescence at the second wavelength tothe fluorescence at the first wavelength.

[0022] The non-oligomerizing fluorescent protein can be a secondnon-oligomerizing tandem fluorescent protein. In addition, the firstnon-oligomerizing tandem fluorescent protein, or the non-oligomerizingfluorescent protein, can contain a targeting sequence, for example, acell compartmentalization domain, which can direct localization of thefluorescent protein in a cell to cytosol, endoplasmic reticulum,mitochondrial matrix, chloroplast lumen, medial trans-Golgi cisternae, alumen of a lysosome, or a lumen of an endosome. In one embodiment, thetargeting sequence is a cell compartmentalization includes amino acidresidues 1 to 81 of human type II membrane-anchored proteingalactosyltransferase, or amino acids 1 to 12 of the presequence ofsubunit IV of cytochrome c oxidase.

[0023] The present invention also relates to a method for determiningwhether a sample contains an enzyme. Such a method can be performed, forexample, by contacting a sample with a tandem non-oligomerizingfluorescent protein; exciting the donor, and determining a fluorescenceproperty in the sample, wherein the presence of the enzyme in the sampleresults in a change in the degree of fluorescence resonance energytransfer. Also provided is a method for determining the activity of anenzyme in a cell. Such a method can be performed, for example, byproviding a cell that expresses a tandem non-oligomerizing tandemfluorescent protein, wherein the peptide linker moiety comprises acleavage recognition amino acid sequence specific for the enzymecoupling the donor and the acceptor, exciting the donor, and determiningthe degree of fluorescence resonance energy transfer in the cell,wherein the presence of enzyme activity in the cell results in a changein the degree of fluorescence resonance energy transfer.

[0024] The present invention also relates to a method for identifyingthe presence of a molecule in a sample. Such a method can be performed,for example, by operatively linking a non-oligomerizing tandemfluorescent protein to the molecule, and detecting fluorescence due tothe non-oligomerizing tandem fluorescent protein in a sample suspectedof containing the molecule, thereby identifying the presence of themolecule in the sample. The molecule can be a polypeptide, for example,an antibody, an enzyme, or a receptor; a polynucleotide; or any othermolecule of interest. The sample can be any sample, including, forexample, a biological sample such as a cell, a tissue sample, or anextract of a cell or a tissue sample. As such, the detecting step can beperformed on an intact cell or tissue sample. The non-oligomerizingtandem fluorescent protein can be operatively linked with the moleculeunder any conditions suitable for linking the protein to the molecule.For example, the protein and molecule can be operatively linked byexpressing a recombinant nucleic acid molecule that encodes thenon-oligomerizing tandem fluorescent protein and the molecule.

[0025] The present invention further relates to a method of identifyingan agent or condition that regulates the activity of an expressioncontrol sequence. Such a method can be performed, for example, byexposing a recombinant nucleic acid molecule, which includes apolynucleotide encoding a non-oligomerizing tandem fluorescent proteinoperatively linked to an expression control sequence, to an agent orcondition suspected of being able to regulate expression of apolynucleotide from the expression control sequence, and detectingfluorescence of the non-oligomerizing tandem fluorescent protein due tosaid exposing, thereby identifying an agent or conditions that regulatesexpression of the expression control sequence. The expression controlsequence can be a transcription regulatory element, for example, apromoter, enhancer, silencer, or insulator, or can be a translationregulatory element, for example, an internal ribosome entry site. Theagent or condition can be any agent or condition, including, forexample, exposure to proteins expressed in a cell.

[0026] The present invention further relates to a method of identifyinga specific interaction of a first molecule and a second molecule. Such amethod can be performed, for example, by contacting the first molecule,which is operatively linked to a donor first non-oligomerizing tandemfluorescent protein, and the second molecule, which is operativelylinked to an acceptor non-oligomerizing fluorescent protein, underconditions that allow a specific interaction of the first molecule andsecond molecule, wherein the first non-oligomerizing tandem fluorescentprotein and the non-oligomerizing fluorescent protein are different;exciting the donor; and detecting fluorescence resonance energy transferfrom the donor to the acceptor, thereby identifying a specificinteraction of the first molecule and the second molecule. In such amethod, the non-oligomerizing fluorescent protein can be a secondnon-oligomerizing tandem fluorescent protein, or can be any othernon-oligomerizing fluorescent protein.

[0027] In one embodiment, the first molecule is a first cellular proteinand the second molecule is a second cellular protein, wherein the firstand second cellular proteins are the same or different. In anotherembodiment, the first molecule is a polynucleotide and the secondmolecule is a polypeptide, for example, a transcription regulatoryelement and a putative transcription factor.

[0028] The present invention also relates to a non-oligomerizingfluorescent protein, which contains at least one mutation that reducesor eliminates the ability of the fluorescent protein to oligomerize. Thenon-oligomerizing fluorescent protein can be derived from anyfluorescent protein that is known to oligomerize, including, forexample, a green fluorescent protein (GFP) such as an Aequorea victoriaGFP, a Renilla reniformis GFP, a Phialidium gregarium GFP; a redfluorescent protein (RFP) such as a Discosoma RFP; or a fluorescentprotein related to a GFP or an RFP. Thus, the non-oligomerizingfluorescent protein can be a cyan fluorescent protein (CFP), or a yellowfluorescent protein (YFP), enhanced GFP (EGFP), an enhanced CFP (ECFP),or an enhanced YFP (EYFP), or a variant of such fluorescent proteins,which can oligomerize but for the presence of one or more mutations thatreduces or eliminates the propensity to oligomerize. Such a mutation canbe, for example, a mutation of one or a combination of amino acidresidues A206, L221 or F223 of Aequorea GFP (SEQ ID NO: 2), or amutation of another fluorescent protein that corresponds to a mutationof A206, L221 or F223 of SEQ ID NO: 2. Such mutations are exemplifiedherein by the mutations A206K, L221K, F223R mutation, or L221K andF223R, of ECFP (SEQ ID NO: 6) and EYFP-V68L/Q69K (SEQ ID NO: 10), whichare spectral variants of Aequorea GFP.

[0029] The present invention also relates to a fusion protein, whichincludes a non-oligomerizing fluorescent protein linked to one or morepolypeptides of interest. The polypeptides of the fusion protein can belinked through peptide bonds, or the non-oligomerizing fluorescentprotein can be linked to the polypeptide of interest through a linkermolecule. A polypeptide of interest can be any polypeptide, including,for example, a peptide tag such as a polyhistidine peptide, or acellular polypeptide such as an enzyme, a G-protein, a growth factorreceptor, or a transcription factor; and can be one of two or moreproteins that can associate to form a complex. In one embodiment, thefusion protein is a tandem non-oligomerizing fluorescent proteinconstruct, which includes a donor non-oligomerizing fluorescent protein,an acceptor non-oligomerizing fluorescent protein, and a peptide linkermoiety coupling said donor and said acceptor, wherein cyclized aminoacids of the donor emit light characteristic of said donor, and whereinthe donor and the acceptor exhibit fluorescence resonance energytransfer when the donor is excited, and the linker moiety does notsubstantially emit light to excite the donor.

[0030] The present invention further relates to a polynucleotide thatencodes an non-oligomerizing fluorescent protein, as well as to a vectorcontaining such a polynucleotide, and a host cell containing apolynucleotide or vector. In addition, the invention relates to arecombinant nucleic acid molecule, which includes a polynucleotideencoding a non-oligomerizing fluorescent protein operatively linked toone or more other polynucleotides. The one or more other polynucleotidescan be, for example, a transcription regulatory element such as apromoter or polyadenylation signal sequence, or a translation regulatoryelement such as a ribosome binding site. Such a recombinant nucleic acidmolecule can be contained in a vector, which can be an expressionvector, and the nucleic acid molecule or the vector can be contained ina host cell.

[0031] The present invention also relates to kits containing one or morecompositions of the invention, for example, one or a plurality ofnon-oligomerizing fluorescent proteins, which can be a portion of afusion protein, or one or a plurality of polynucleotides that encode theproteins. A kit of the invention also can contain one or a plurality ofrecombinant nucleic acid molecules, which encode, in part,non-oligomerizing fluorescent proteins, which can be the same ordifferent, and further include, for example, an operatively linkedsecond polynucleotide containing or encoding a restriction endonucleaserecognition site or a recombinase recognition site, or any polypeptideof interest.

[0032] The present invention further relates to a method for identifyingthe presence of a molecule in a sample. Such a method can be performed,for example, by linking a non-oligomerizing fluorescent protein to themolecule, and detecting fluorescence due to the non-oligomerizingfluorescent protein in a sample suspected of containing the molecule,thereby identifying the presence of the molecule in the sample. Themolecule to be detected can be any molecule, including, for example, apolypeptide such as an antibody, an enzyme, or a receptor, or apolynucleotide. In addition, the sample can be any sample, including abiological sample such as a cell, which can be a cell in culture or acell isolated from an organism, a tissue sample, or an extract of a cellor a tissue sample. In one embodiment, the method is performed using anintact cell or tissue sample, wherein the presence of a molecule ofinterest in living cells can be identified.

[0033] Linking of the non-oligomerizing fluorescent protein to themolecule can be performed using an linkage that is stable under theconditions to which the polypeptide-molecule complex is to be exposed,and can be performed using a chemical reaction or can result ofexpression of a recombinant nucleic acid molecule encoding the linkedcomplex. Thus, linking can be performed by contacting thenon-oligomerizing fluorescent protein with the molecule under conditionssuitable for linking the protein to the molecule, such conditionsdepending, for example, on the chemical nature of the molecule and thetype of linkage desired, which can be a direct linkage or can bemediated by a linker moiety. Where the molecule is a polypeptide,linking can be performed by expressing a recombinant nucleic acidmolecule comprising a polynucleotide encoding the non-oligomerizingfluorescent protein operatively linked to a polynucleotide encoding themolecule.

[0034] The present invention also relates to a method of identifying anagent or condition that regulates the activity of an expression controlsequence. Such a method can be performed, for example, by exposing arecombinant nucleic acid molecule, which includes a polynucleotideencoding a non-oligomerizing fluorescent protein operatively linked toan expression control sequence, to an agent or condition suspected ofbeing able to regulate expression of a polynucleotide from theexpression control sequence, and detecting fluorescence of thenon-oligomerizing fluorescent protein due to such exposure, therebyidentifying an agent or conditions that regulates expression of theexpression control sequence. The expression control sequence can be anysuch sequence, including, for example, a transcription regulatoryelement such as a promoter or a translation regulatory element such as aribosome binding site. In addition, the agent can be any agent,including, for example, a peptide, polynucleotide, small organicmolecule or the like. Similarly, the condition can be any condition,including, for example, exposure to proteins expressed in a cell and,therefore, the method can be used to identify a transcription factor, atranslation factor, or the like, including tissue-specific factors.

[0035] The present invention also relates to a method of identifying aspecific interaction of a first molecule and a second molecule. Such amethod can be performed, for example, by contacting the first molecule,which is linked to a donor first non-oligomerizing fluorescent protein,and the second molecule, which is linked to an acceptor secondnon-oligomerizing fluorescent protein, under conditions that allow aspecific interaction of the first molecule and second molecule; excitingthe donor; and detecting fluorescence resonance energy transfer from thedonor to the acceptor, thereby identifying a specific interaction of thefirst molecule and the second molecule. The first and second moleculecan be cellular proteins, which are the same or different, or can be apolynucleotide and a polypeptide, thus providing, for example, a meansto identify proteins that specifically interact such as proteinsinvolved in transducing an intracellular signal, or to identify atranscription regulatory element that specifically binds a transcriptionfactor.

[0036] The present invention also relates to a method for determiningwhether a sample contains an enzyme. Such a method can be performed, forexample, by contacting a sample with a tandem non-oligomerizingfluorescent protein construct of the invention; exciting the donor, anddetermining a fluorescence property in the sample, wherein the presenceof an enzyme in the sample results in a change in the degree offluorescence resonance energy transfer. Similarly, the present inventionrelates to a method for determining the activity of an enzyme in a cell.Such a method can be performed, for example, providing a cell thatexpresses a tandem non-oligomerizing fluorescent protein construct,wherein the peptide linker moiety comprises a cleavage recognition aminoacid sequence specific for the enzyme coupling the donor and theacceptor; exciting said donor, and determining the degree offluorescence resonance energy transfer in the cell, wherein the presenceof enzyme activity in the cell results in a change in the degree offluorescence resonance energy transfer.

[0037] The present invention further relates to a method for determiningthe pH of a sample. Such a method can be performed, for example, bycontacting the sample with a first non-oligomerizing fluorescentprotein, wherein the emission intensity of the first non-oligomerizingfluorescent protein changes as pH varies between pH 5 and pH 10;exciting the indicator; and determining the intensity of light emittedby the first non-oligomerizing fluorescent protein at a firstwavelength, wherein the emission intensity of the firstnon-oligomerizing fluorescent protein indicates the pH of the sample.The first non-oligomerizing fluorescent protein useful in this method,or in any method of the invention, can have an amino acid sequence ofSEQ ID NO: 2, or a sequence substantially identical thereto, forexample, having the mutations S65G/S72A/T203Y/H231L with respect to SEQID NO: 2, or having the mutations S65G/V68L/Q69K/S72A/T203Y/H231L withrespect to SEQ ID NO: 2; or having the mutationsK26R/F64L/S65T/Y66W/N146I/M153T/V163A/N164H/H231L with respect to SEQ IDNO: 2; or any of the above non-oligomerizing fluorescent protein furtherhaving a mutation corresponding to H148G or H148Q with respect to SEQ IDNO: 2.

[0038] The sample used in a method for determining the pH of a samplecan be any sample, including, for example, a biological tissue sample,or a cell or a fraction thereof. In addition, the method can furtherinclude contacting the sample with a second non-oligomerizingfluorescent protein, wherein the emission intensity of the secondnon-oligomerizing fluorescent protein changes as pH varies from 5 to 10,and wherein the second non-oligomerizing fluorescent protein emits at asecond wavelength that is distinct from the first wavelength; excitingthe second non-oligomerizing fluorescent protein; determining theintensity of light emitted by the second non-oligomerizing fluorescentprotein at the second wavelength; and comparing the fluorescence at thesecond wavelength to the fluorescence at the first wavelength. The first(or second) non-oligomerizing fluorescent protein can include atargeting sequence, for example, a cell compartmentalization domain sucha domain that targets the non-oligomerizing fluorescent protein in acell to the cytosol, the endoplasmic reticulum, the mitochondrialmatrix, the chloroplast lumen, the medial trans-Golgi cisternae, a lumenof a lysosome, or a lumen of an endosome. For example, the cellcompartmentalization domain can include amino acid residues 1 to 81 ofhuman type II membrane-anchored protein galactosyltransferase, or aminoacid residues 1 to 12 of the presequence of subunit IV of cytochrome coxidase.

BRIEF DESCRIPTION OF THE DRAWING

[0039]FIG. 1 illustrates the tetrameric form of DsRed (PDBidentification code 1G7K). The A-C and B-D interfaces are equivalent, asare the A-B and C-D interfaces. The labeling of the subunits isarbitrary and, therefore, the convention used in this FIGURE differsfrom 1G7K, but is consistent with the PDB submission 1GGK.

DETAILED DESCRIPTION OF THE INVENTION

[0040] The present invention provides non-oligomerizing fluorescentproteins, which are derived from fluorescent proteins that canoligomerize. As disclosed herein, a non-oligomerizing fluorescentprotein of the invention can be derived from a naturally occurringfluorescent protein or from a spectral variant or mutant thereof, andcontains at least one mutation that reduces or eliminates the ability ofthe fluorescent protein to oligomerize.

[0041] A non-oligomerizing fluorescent protein of the invention can bederived from any fluorescent protein that is known to oligomerize,including, for example, a green fluorescent protein (GFP) such as anAequorea victoria GFP, a Renilla reniformis GFP, a Phialidium gregariumGFP; a red fluorescent protein (RFP) such as a Discosoma RFP; or afluorescent protein related to a GFP or an RFP. Thus, thenon-oligomerizing fluorescent protein can be a cyan fluorescent protein(CFP), a yellow fluorescent protein (YFP), an enhanced GFP (EGFP; SEQ IDNO: 4), an enhanced CFP (ECFP; SEQ ID NO: 6), an enhanced YFP (EYFP; SEQID NO: 8), a DsRed fluorescent protein (SEQ ID NO: 12), or a mutant orvariant of such fluorescent proteins.

[0042] As disclosed herein, the propensity of the non-oligomerizingfluorescent proteins of the invention to oligomerize is reduced oreliminate. In one embodiment, the propensity of a non-oligomerizingfluorescent protein to oligomerize is reduced or eliminated due tooperatively linking a first monomer of a fluorescent protein to at leasta second monomer of the fluorescent protein, thereby forming anintramolecular ‘dimer’, ‘trimer’ or the like. Such operatively linkedhomopolymers, which are referred to herein as “non-oligomerizing tandemdimers,” have a substantially reduced ability to form intermolecularoligomers. Such non-oligomerizing tandem fluorescent proteins areexemplified herein by two monomers of DsRed (SEQ ID NO:12) operativelylinked by a peptide linker (SEQ ID NO:26), and by two monomers of amutant DsRed, which has an amino acid sequence of SEQ ID NO:12, andincluding an I125R mutation, operatively linked by the peptide linker ofSEQ ID NO:26.

[0043] In another embodiment, the propensity of a non-oligomerizingfluorescent protein to oligomerize is reduced or eliminated due to thepresence of one or more mutations in the fluorescent protein. Suchmutations are exemplified by a mutation of one or a combination of aminoacid residues A206, L221 or F223 of Aequorea GFP (SEQ ID NO: 2), or amutation of another fluorescent protein that corresponds to a mutationof A206, L221 or F223 of SEQ ID NO: 2, for example, by the mutationsA206K, L221 K, F223R of GFP (SEQ ID NO:2), or by the mutations L221K andF223R of ECFP (SEQ ID NO: 6) and EYFP-V68L/Q69K (SEQ ID NO: 10), whichare spectral variants of Aequorea GFP.

[0044] As used herein, the term “non-oligomerizing tandem fluorescentprotein” refers to a composition of two or more monomers of afluorescent protein that are operatively linked and that exhibit acharacteristic fluorescence emission spectrum and fluorescenceexcitation spectrum. As disclosed herein, the intramolecular‘oligomerization’ characteristic of a non-oligomerizing tandemfluorescent protein of the invention reduces or eliminates thepropensity of fluorescent protein to undergo intermolecularoligomerization.

[0045] The term “non-oligomerizing fluorescent protein” is used morebroadly herein to refer to fluorescent proteins that have been modifiedsuch that they have a reduced propensity to oligomerize as compared to acorresponding unmodified fluorescent protein. As such, unlessspecifically indicated otherwise, the term “non-oligomerizingfluorescent protein” encompasses non-oligomerizing tandem fluorescentproteins, as well as fluorescent proteins that contain one or moremutations that reduce or eliminate the propensity of the fluorescentprotein to oligomerize.

[0046] The term “tandem non-oligomerizing fluorescent protein” is usedherein to refer to a composition containing two different fluorescentproteins, including a donor fluorescent protein operatively linked to anacceptor fluorescent protein, wherein at least one of the fluorescentproteins is a non-oligomerizing fluorescent protein. As such, withrespect to its fluorescent protein components, a “tandemnon-oligomerizing fluorescent protein” can be analogized to aheteropolymer, whereas a “non-oligomerizing tandem fluorescent protein”can be analogized to a homopolymer.

[0047] Aequorea GFP is widely used in cell biology as a protein modulethat can be fused to host proteins to make the latter fluorescent(Tsien, Ann. Rev. Biochem. 67:509-544, 1998, which is incorporatedherein by reference). For example, GFP is commonly used to characterizesubcellular localization and trafficking properties of proteins, towhich the GFP is fused. In addition, spectral variants of GFP, includingCFP and YFP and variants thereof have been used to measure theassociative properties of host proteins by fluorescence resonance energytransfer (FRET). FRET between CFP and YFP also has been exploited tocreate biosensors for calcium ion, and to determine the associativeproperties of growth factor receptors and G protein-coupled receptors.

[0048] The GFP spectral mutants, CFP and YFP and variants thereof suchas ECFP (SEQ ID NO: 6) and EYFP-V68L/Q69K (SEQ ID NO: 10), have most ofthe desirable properties required of good FRET partners, except thatthese proteins exhibit homoaffinity and form dimers. As such, GFP andits spectral variants, show distinct tendencies to dimerize in somecrystal structures, in solution, and in many conditions inside cells.Such dimerization means that host proteins fused to a GFP (or variant)can be induced to dimerize, thereby perturbing their functions andresulting in artifacts when FRET between different colors of GFPspectral variants is used to assess protein-protein interaction.Accordingly, it would be desirable to identify mutations that caneliminate the tendency of all colors of GFP spectral variants todimerize, without having any deleterious effects on other properties ofthe fluorescent proteins. As disclosed herein, the mutations, A206K,L221K and F223R, either alone or in combinations, in ECFP (SEQ ID NO: 6)and EYFP-V68L/Q69K (SEQ ID NO: 10) reduce or eliminate the propensity ofGFP and its spectral variants to dimerize. Thus, except wheredimerization is positively desired, one or more of these mutations canbe routinely incorporated into a GFP variant, thereby reducing oreliminating its ability to induce artifactual dimerization.

[0049] Although Aequorea GFP has proven to be a tool of great value tocell biologists, the propensity of GFP to dimerize at relatively lowexpression levels in cells has limited the development of new and betterassays, particularly assays for the localization of host proteins andthe determination of their associative properties. The present inventionprovides a means to substantially reduce or eliminate the ability offluorescent proteins such as GFP to oligomerize, thereby solving theproblems associated therewith, and allowing the development of assaysthat could not previously be performed.

[0050] Another limitation is that, while GFP variants with blue, cyan,and yellowish green emissions have been engineered, all have emissionmaxima shorter than 529 nm. Recently, polynucleotides encoding sixanthozoan (coral) fluorescent proteins having 26% to 30% identity toAequorea GFP (SEQ ID NO: 2) were cloned by Matz et al. (NatureBiotechnol. 17:969-973, 1999, which is incorporated herein byreference). Although most of the coral fluorescent proteins had emissionmaxima within the range covered by GFP or its variants, one coralprotein, drFP583 (“DsRed”; SEQ ID NO: 12), which was isolated from a redportion of a Discosoma species, had excitation and emission maxima at558 and 583 nm, respectively, the longest yet reported for a wild typespontaneously fluorescent protein (Matz et al., supra, 1999). Despitethe relatively modest sequence identity to GFP, enough sequencesimilarity was conserved to suggest that the coral proteins would form11-stranded θ-barrels, similar to that of GFP. In addition, the twoimportant residues contributing to the chromophore of GFP, Tyr66 andGly67, and some of the important polar residues contacting thechromophore such as Arg96 and Glu222, were conserved in the coralproteins. In DsRed, the amino acids corresponding to these GFP residuesare numbered Tyr67, Gly68, Arg95, and Glu215, respectively, andadditional amino acids that can be involved in oligomerization can beidentified using X-ray crystallography methods (see Example 3).

[0051] The cloning of a red fluorescent protein (DsRed) from Discosomaraised a great deal of interest due to its tremendous potential as atool for the advancement of cell biology. However, a carefulinvestigation of the properties of this protein revealed severalproblems that would preclude DsRed from being as widely accepted as theAequorea GFP and its blue, cyan, and yellow variants, which have foundwidespread use as both genetically encoded indicators for tracking geneexpression and as donor/acceptor pairs for fluorescence resonance energytransfer (FRET). Extending the spectrum of available colors to redwavelengths would provide a distinct new label for multicolor trackingof fusion proteins and together with GFP would provide a new FRETdonor/acceptor pair that would be superior to the currently preferredcyan/yellow pair.

[0052] The two most pressing problems with the 28 kDa DsRed are itsstrong tendency to oligomerize and its slow maturation. A variety oftechniques have been used to determine that DsRed is an obligatetetramer both in vitro and in vivo. For numerous reasons, the oligomericstate of DsRed is problematic for applications in which it is fused to aprotein of interest in order to monitor trafficking or interactions ofthe latter. Using purified protein, it was shown that DsRed requiresgreater than 48 hours to reach >90% of its maximal red fluorescence (seebelow). During the maturation process, a green intermediate initiallyaccumulates and is slowly converted to the final red form. However, theconversion of the green component does not proceed to completion andthus a fraction of aged DsRed remains green. The primary disadvantage ofthe incomplete maturation is an excitation spectrum that extends wellinto the green wavelengths due to energy transfer between the green andred species within the tetramer. This is a particularly serious problemdue to overlap with the excitation spectra of potential FRET partnerssuch as GFP.

[0053] The original report of the cloning of DsRed provided an in vivoapplication marking the fates of Xenopus blastomeres after 1 week ofdevelopment (Matz et al., supra, 1999). As disclosed herein, DsRed hasbeen characterized with respect to the time the red fluorescence takesto appear, the pH sensitivity of the chromophore, how strongly thechromophore absorbs light and fluoresces, how readily the proteinphotobleaches, and whether the protein normally exists as a monomer oran oligomer in solution (see Example 2). The results demonstrate thatDsRed provides a useful complement to or alternative for GFP and itsspectral mutants. In addition, DsRed mutants that are non-fluorescent orthat are blocked or slowed in converting from green to red emission werecharacterized, including mutants in which the eventual fluorescence issubstantially red-shifted from wild type DsRed (see Example 2; see,also, Baird et al., Proc. Natl. Acad. Sci., USA 97:11984-11989, 2000;Gross et al., Proc. Natl. Acad. Sci., USA 97:11990-11995, 2000, each ofwhich is incorporated herein by reference).

[0054] As disclosed herein, mutations were introduced into DsRed similarto those introduced into the GFP spectral variants, and DsRed mutantshaving reduced oligomerization activity were identified, including, forexample, a DsRed-I125R mutant of DsRed as set forth in SEQ ID NO: 12(see Example 3). The strategy for producing the DsRed mutants involvedintroducing mutations in DsRed that were predicted to interfere with thedimer interfaces (A-B or A-C, see FIG. 1) and thus prevent formation ofthe tetramer. This strategy resulted in the production of DsRed mutantsthat had a reduced propensity to form tetramers by disrupting the A-Cinterface, for example, using the single replacement of isoleucine 125with an arginine (I125R).

[0055] The A-B interface proved to be more resilient, and mutations thatpotentially could disrupt this interface were ineffective and resultedin non-fluorescent proteins. As disclosed herein, a novel approach wasused to overcome the intermolecular oligomerization propensity of DsRedby linking the C-terminus of the A subunit to the N-terminus of the Bsubunit through a flexible tether to produce tandem dimers. Based on thecrystal structure of DsRed, an 18 residue linker (Whitlow et al., Prot.Eng. 6:989-995, 1993, which is incorporated herein by reference) waspredicted to be long enough to extend from the C-terminus of the Asubunit to the N-terminus of the B subunit (about 30 Å), but not fromthe N-terminus of the C subunit (greater than 70 Å). As such,‘oligomerization’ in the tandem dimers is intramolecular, i.e., thetandem dimer of DsRed (tDsRed), for example, is encoded by a singlepolypeptide chain. Furthermore, a combination of tDsRed with the I125Rmutant (tDsRed-I125R) resulted in a monomeric red fluorescent proteinthat effectively solved the oligomer problem (Example 4). It should berecognized that this strategy can be generally applied to any proteinsystem in which the distance between the N-terminus of one protein andthe C-terminus of a dimer partner is known, such that a linker havingthe appropriate length can be used to operatively link the monomers. Inparticular, this strategy can be useful for other modifying otherfluorescent proteins that have interesting spectral properties, but formobligate dimers that are difficult to disrupt using the targetedmutagenesis method disclosed herein.

[0056] The availability of a wide range of variously-colored “spectralmutants” of GFP has provided a potential means for monitoring theassociative properties of proteins via FRET. FRET is a quantummechanical phenomenon of radiation-less energy transfer between twofluorophores, that is dependent on the proper spectral overlap of adonor and an acceptor, their distance from each other, and the relativeorientation of the chromophores' transition dipoles. Using standardmolecular biology technology, fusions can be generated between proteinsof interest and spectral mutants of fluorescent proteins, which can thenserve effectively as donor and acceptor FRET partners. As indicatedabove, the GFP spectral mutants have most of the requisite properties toserve as useful FRET partners, except for their homoaffinity andpropensity for dimerization. Thus, while the number of FRET-based assaysusing GFP and its variants is increasing (see, for example, Mitra etal., Gene 173:13-17, 1996; Hartman and Vale, Science 286:782-785, 1999;Zacharias et al., Curr. Opin. Neurobiol. 10:416-421, 2000), thepropensity of the GFP-related fluorescent proteins to associate witheach other can complicate characterization of protein associationsreported by FRET, which should be due solely to interactions of theproteins with no participation from the fluorophore to which they arelinked.

[0057] FRET assays using GFP spectral variants can fail becausedimerization can mask or mimic host protein interactions such that thedata cannot be interpreted. Changes in FRET can be masked, for example,when dimerization of a CFP or YFP supersedes or prevents aconformational change of an intervening peptide or protein, or whendissociation of two or more host proteins is not allowed or is impededdue to dimerization of the fluorescent proteins. Similarly, if a CFP andYFP are present in a single fusion protein, a dimer interaction betweenthese proteins can result, eliminating the ability to detect a changethat may have occurred within a single fusion protein, similar to thehypothesized oligomerization of chameleons. Thus, situations wherechanges in FRET are mimicked can occur when dimerization of the GFPs orGFP spectral mutants mimic an interaction that otherwise is believed tobe occurring between two host proteins.

[0058] In addition to interfering with FRET analysis, oligomerization offluorescent proteins such as DsRed, GFP and its variants causes otherproblems that limit its usefulness. For example, another important andcommon application of these proteins is as a fluorescent label forobserving, in living cells, the subcellular localization or distributionof proteins to which the fluorescent protein has been fused. Dependingon the localization and naturally-occurring oligomeric state of theprotein to which the fluorescent protein is fused, the fluorescentproteins can reach a local concentration in a cell in excess of thatrequired for dimerization, thus altering the spatial distribution orfunction of its fusion partner.

[0059] It is difficult to determine in advance whether any of theproblems associated with dimerization of fluorescent marker proteinswill invalidate the results of a particular assay. However, mimicking ofan intramolecular interaction where none exists, for example, FRETbetween a CFP and a YFP fused to two separate proteins, can occur whenthe fluorescent proteins are targeted to various subcellular locationssuch as the plasma membrane (PM), or even when expressed free in thecytoplasm (Miyawaki and Tsien, Meth. Enzymol. 327:472-500, 2000, whichis incorporated herein by reference). Since such artifacts can bedifficult to detect and prove, it would a great advantage ifdimerization of the fluorescent proteins can be avoided. As disclosedherein, the present invention provides a means to substantially reduceor eliminate the propensity of fluorescent proteins to dimerize, therebyenabling accurate monitoring of the associative properties anddistributions of host proteins in a cell, including erroneous FRETcaused by fluorescent protein oligomerization, as well as other problemssuch as protein localization associated with such oligomerization.

[0060] The crystal structures of GFP and several of its variants havebeen solved (see, for example, Ormo et al., Science 273:1392-1395, 1996;Yang et al., Nature Biotechnol. 14:1246-1251, 1996; Wachter et al.,Biochemistry 36:9759-9765, 1997; Palm et al., Nature Struct. Biol.4:361-365,1997, each of which is incorporated herein by reference).Depending on the experimental conditions used to form the crystal, thecrystallographic unit cell is a head-to-tail, side-by-side dimer(Phillips, In “Green Fluorescent Protein: Properties, Applications andProtocols” (eds. Chalfie and Kain 1998), pages 77-96, which isincorporated herein by reference; see, also, Yang et al, supra, 1996;Tsien, supra, 1998). In order to form crystals, GFP must be veryconcentrated. As such, the structure of GFP in a crystal may notrepresent the state of the protein in solution. However, other lines ofevidence indicate that GFP and its variants can form dimers in solutionand that dimerization can occur at the concentrations and conditionsthat commonly exist in a cell-biological context (Ward et al., In “GreenFluorescent Protein: Properties, Applications and Protocols” (eds.Chalfie and Kain 1998), pages 45-75, which is incorporated herein byreference; see, also, Phillips, supra, 1998).

[0061] Contact sites identified in one crystal structure included a coreof hydrophobic side chains from each of the two monomers and potentiallymany hydrophilic contacts (Yang et al., supra, 1996). This patch ofhydrophobic side chains has been suggested to play a role in theassociation of GFP with the Ca²⁺-sensitive photoprotein, aequorin in thejellyfish. Residues A206, L221 and F223 appeared to be reasonablecandidates for creating the contacts between monomers when GFP is insolution or expressed exogenously in cells (Yang et al., supra, 1996;Phillips, supra, 1998). In order to determine whether one or more ofthese residues affect dimerization under physiological conditions,mutations that substituted amino acid residues having positively chargedside chains were introduced and the interactions between the mutagenizedmonomers was examined. A quantitative determination of dimer affinitywas made by subjecting highly purified ECFP (SEQ ID NO: 6) andEYFP-V68L/Q69K (SEQ ID NO: 10), and “dimer mutants” derived therefrom,to analytical ultracentrifugation, which can very accurately determinethe degree of association between self associating proteins (McRorie andVoelker, In “Self-associating systems in the analytical ultracentrifuge(Beckman Instruments 1993)). Similarly, ECFP (SEQ ID NO: 6) andEYFP-V68L/Q69K (SEQ ID NO: 10) targeted to the plasma membrane, anddimer mutants derived from these GFP variants, were used in cellbiological experiments designed specifically to determine theself-associative behavior of the various proteins.

[0062] As disclosed herein, amino acid residues A206, L221 and F223 of aGFP (see, for example, SEQ ID NO: 2) are sufficient to inducedimerization of GFP and spectral variants thereof at relatively lowconcentrations in solution and in living cells, and mutations of A206,L221 and F223, alone or in combination, to positively-charged residuessubstantially reduced or eliminated the interaction of the monomers insolution and in living cells. Since ECFP (SEQ ID NO: 6) andEYFP-V68L/Q69K (SEQ ID NO: 10), and virtually all other GFP-relatedmutants, have the same residue composition at these three positions asthe wild type GFP (Prasher et al., Gene 111:229-233, 1992, which isincorporated herein by reference), the present results indicate thatcorresponding mutations in other fluorescent proteins havingsubstantially the same general structure, including GFP spectralvariants and the RFP, DsRed, similarly can reduce or eliminate theability of the proteins to oligomerize (see Examples 1 and 3).

[0063] Other Aequorea GFP-related fluorescent proteins that can bemodified according to a method of the invention so as to reduce oreliminate the propensity to oligomerize are well known in the art, andare exemplified by those having the mutations F64L,S65T, Y66W, F99S, orVI 63A, wherein the amino acid residues are referred to with respect toSEQ ID NO: 2, including variants thereof as disclosed in InternationalPubl. No. WO 00/71565 A2, published Nov. 30, 2000, which is incorporatedherein by reference. The numbering of the GFP amino acids as referred toherein conforms to that in native Aequorea GFP (SEQ ID NO: 2), whereinthe first serine is amino acid number 2 even if a valine (amino acid no.1a) has been inserted to optimize ribosome initiation. For example, F64Lrefers to a substitution of leucine for phenylalanine at amino acidposition 64 following the initiating methionine.

[0064] Examples of GFP spectral variants in addition to CFP and YFP,include, for example, enhanced GFP (EGFP; SEQ ID NO: 4;F64L/S65T/H231L); EYFP (SEQ ID NO: 8; S65G/S72A/T203Y/H231L);EYFP-V68L/Q69K (SEQ ID NO: 10; S65G/V68L/Q69K/S72A/T203Y/H231L); ECFP(SEQ ID NO: 6; K26R/F64L/S65T/Y66W/N146I/M153T/V163A/N164H/H231L), andthe like; and variants of these GFP-related fluorescent proteins havingthe mutation H148G or H148Q, wherein the indicated mutations are withrespect to SEQ ID NO: 2 (see International Publ. No. WO 00/71565 A2,supra, 2000). Additional examples of fluorescent proteins that can bemodified to reduce or eliminate the propensity to oligomerize includeDsRed and variants thereof, which, as disclosed herein, can havedesirable fluorescent characteristics as compared to native DsRed (seeExamples 2 and 3), yellow fluorescent protein from Vibrio fischeristrain Y-1, Peridinin-chlorophyll a binding protein from thedinoflagellate Symbiodinium phycobiliproteins from marine cyanobacteriasuch as Synechococcus, for example, phycoerythrin and phycocyanin, oroat phytochromes from oat reconstructed with phycoerythrobilin (seeBaldwin, Biochemistry 29:5509-5515, 1990; Morris et al., Plant Mol.Biol. 24:673-677, 1994; Wilbanks et al., J. Biol. Chem. 268:1226-1235,1993; Li et al., Biochemistry 34:7923-7930, 1995; Murphy and Lagarias,Curr. Biol. 7: 870-876, 1997, each of which is incorporated herein byreference).

[0065] Unless specifically indicated otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood by those of ordinary skill in the art to which this inventionbelongs. In addition, any method or material similar or equivalent to amethod or material described herein can be used in the practice thepresent invention. For purposes of the present invention, the followingterms are defined.

[0066] The term “nucleic acid molecule” or “polynucleotide” refers to adeoxyribonucleotide or ribonucleotide polymer in either single-strandedor double-stranded form, and, unless specifically indicated otherwise,encompasses polynucleotides containing known analogs of naturallyoccurring nucleotides that can function in a similar manner as naturallyoccurring nucleotides. It will be understood that when a nucleic acidmolecule is represented by a DNA sequence, this also includes RNAmolecules having the corresponding RNA sequence in which “U” (uridine)replaces “T” (thymidine).

[0067] The term “recombinant nucleic acid molecule” refers to anon-naturally occurring nucleic acid molecule containing two or morelinked polynucleotide sequences. A recombinant nucleic acid molecule canbe produced by recombination methods, particularly genetic engineeringtechniques, or can be produced by a chemical synthesis method. Arecombinant nucleic acid molecule can encode a fusion protein, forexample, a non-oligomerizing fluorescent protein of the invention linkedto a polypeptide of interest. The term “recombinant host cell” refers toa cell that contains a recombinant nucleic acid molecule. As such, arecombinant host cell can express a polypeptide from a “gene” that isnot found within the native (non-recombinant) form of the cell.

[0068] Reference to a polynucleotide “encoding” a polypeptide meansthat, upon transcription of the polynucleotide and translation of themRNA produced therefrom, a polypeptide is produced. The encodingpolynucleotide is considered to include both the coding strand, whosenucleotide sequence is identical to an mRNA, as well as itscomplementary strand. It will be recognized that such an encodingpolynucleotide is considered to include degenerate nucleotide sequences,which encode the same amino acid residues. Nucleotide sequences encodinga polypeptide can include polynucleotides containing introns as well asthe encoding exons.

[0069] The term “expression control sequence” refers to a nucleotidesequence that regulates the transcription or translation of apolynucleotide or the localization of a polypeptide to which to which itis operatively linked. Expression control sequences are “operativelylinked” when the expression control sequence controls or regulates thetranscription and, as appropriate, translation of the nucleotidesequence (i.e., a transcription or translation regulatory element,respectively), or localization of an encoded polypeptide to a specificcompartment of a cell. Thus, an expression control sequence can be apromoter, enhancer, transcription terminator, a start codon (ATG), asplicing signal for intron excision and maintenance of the correctreading frame, a STOP codon, a ribosome binding site, or a sequence thattargets a polypeptide to a particular location, for example, a cellcompartmentalization signal, which can target a polypeptide to thecytosol, nucleus, plasma membrane, endoplasmic reticulum, mitochondrialmembrane or matrix, chloroplast membrane or lumen, medial trans-Golgicistemae, or a lysosome or endosome. Cell compartmentalization domainsare well known in the art and include, for example, a peptide containingamino acid residues 1 to 81 of human type II membrane-anchored proteingalactosyltransferase, or amino acid residues 1 to 12 of the presequenceof subunit IV of cytochrome c oxidase (see, also, Hancock et al., EMBOJ. 10:4033-4039, 1991; Buss et al., Mol. Cell. Biol. 8:3960-3963, 1988;U.S. Pat. No. 5,776,689, each of which is incorporated herein byreference).

[0070] The term “operatively linked” also is used in reference to themonomeric fluorescent protein components of a non-oligomerizing tandemfluorescent protein of the invention, as well as to components of afusion protein comprising a non-oligomerizing fluorescent protein,including a non-oligomerizing tandem fluorescent protein, andpolypeptide of interest. With respect to a non-oligomerizing tandemfluorescent protein, the term “operatively linked” means that thenon-oligomerizing tandem fluorescent protein has a characteristicfluorescence emission and excitation spectra. The fluorescence emissionand excitation spectra of the non-oligomerizing tandem fluorescentprotein can be the same as the spectra of the monomeric form of thefluorescent protein comprising the non-oligomerizing tandem fluorescentprotein, or the spectra can be different from those of the monomericfluorescent protein. With respect to a fusion protein comprising anon-oligomerizing fluorescent protein, the term “operatively linked”means that the polypeptide components of the fusion protein are linkedsuch that each maintains its function, including the fluorescencecharacteristics of the non-oligomerizing fluorescent protein and anyfunction characteristic or of particular interest of the polypeptidelinked thereto. The term “operatively linked” similarly is used hereinto refer to the components of a tandem non-oligomerizing fluorescentprotein of the invention, which comprises a first non-oligomerizingfluorescent protein, which can be a non-oligomerizing tandem fluorescentprotein, and a second fluorescent protein, which can, but need not be anon-oligomerizing fluorescent protein, wherein the first fluorescentprotein and second fluorescent protein are linked such that eachmaintains its fluorescence activity.

[0071] The term “oligomer” refers to a complex formed by the specificinteraction of two or more polypeptides. A “specific interaction” or“specific association” is one that is relatively stable under specifiedconditions, for example, physiologic conditions. Reference to a“propensity” of proteins to oligomerize indicates that the proteins canform dimers, trimers, tetramers, or the like under specified conditions.Generally, fluorescent proteins such as GFPs and DsRed have a propensityto oligomerize under physiologic conditions although, as disclosedherein, fluorescent proteins also can oligomerize, for example, under pHconditions other than physiologic conditions. The conditions under whichfluorescent proteins oligomerize or have a propensity to oligomerize canbe determined using well known methods as disclosed herein (see Examples1 and 3) or otherwise known in the art.

[0072] The term “probe” refers to a substance that specifically binds toanother substance (a “target”). Probes include, for example, antibodies,polynucleotides, receptors and their ligands, and generally can belabeled so as to provide a means to identify or isolate a molecule towhich the probe has specifically bound. The term “label” refers to acomposition that is detectable with or without the instrumentation, forexample, by visual inspection, spectroscopy, or a photochemical,biochemical, immunochemical or chemical reaction. Useful labels include,for example, phosphorus-32, a fluorescent dye, a fluorescent protein, anelectron-dense reagent, an enzymes (such as is commonly used in anELISA), a small molecule such as biotin, digoxigenin, or other haptensor peptide for which an antiserum or antibody, which can be a monoclonalantibody, is available. It will be recognized that a non-oligomerizingfluorescent protein of the invention, which is itself a detectableprotein, can nevertheless be labeled so as to be detectable by a meansother than its own fluorescence, for example, by incorporating aradionuclide label or a peptide tag into the protein so as tofacilitate, for example, identification of the protein during itsexpression and isolation of the expressed protein, respectively. A labeluseful for purposes of the present invention generally generates ameasurable signal such as a radioactive signal, fluorescent light,enzyme activity, and the like, either of which can be used, for example,to quantitate the amount of the non-oligomerizing fluorescent protein ina sample.

[0073] The term “nucleic acid probe” refers to a polynucleotide thatbinds to a specific nucleotide sequence or sub-sequence of a second(target) nucleic acid molecule. A nucleic acid probe generally is apolynucleotide that binds to the target nucleic acid molecule throughcomplementary base pairing. It will be understood that a nucleic acidprobe can specifically bind a target sequence that has less thancomplete complementarity with the probe sequence, and that thespecificity of binding will depend, in part, upon the stringency of thehybridization conditions. A nucleic acid probes can be labeled as with aradionuclide, a chromophore, a lumiphore, a chromogen, a fluorescentprotein, or a small molecule such as biotin, which itself can be bound,for example, by a streptavidin complex, thus providing a means toisolate the probe, including a target nucleic acid molecule specificallybound by the probe. By assaying for the presence or absence of theprobe, one can detect the presence or absence of the target sequence orsub-sequence. The term “labeled nucleic acid probe” refers to a nucleicacid probe that is bound, either directly or through a linker molecule,and covalently or through a stable non-covalent bond such as an ionic,van der Waals or hydrogen bond, to a label such that the presence of theprobe can be identified by detecting the presence of the label bound tothe probe.

[0074] The term “polypeptide” or “protein” refers to a polymer of two ormore amino acid residues. The terms apply to amino acid polymers inwhich one or more amino acid residue is an artificial chemical analogueof a corresponding naturally occurring amino acid, as well as tonaturally occurring amino acid polymers. The term “recombinant protein”refers to a protein that is produced by expression of a nucleotidesequence encoding the amino acid sequence of the protein from arecombinant DNA molecule.

[0075] The term “isolated” or “purified” refers to a material that issubstantially or essentially free from components that normallyaccompany the material in its native state in nature. Purity orhomogeneity generally are determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis, high performanceliquid chromatography, and the like. A polynucleotide or a polypeptideis considered to be isolated when it is the predominant species presentin a preparation. Generally, an isolated protein or nucleic acidmolecule represents greater than 80% of the macromolecular speciespresent in a preparation, often represents greater than 90% of allmacromolecular species present, usually represents greater than 95%, ofthe macromolecular species, and, in particular, is a polypeptide orpolynucleotide that purified to essential homogeneity such that it isthe only species detected when examined using conventional methods fordetermining purity of such a molecule.

[0076] The term “naturally-occurring” is used to refer to a protein,nucleic acid molecule, cell, or other material that occurs in nature.For example, a polypeptide or polynucleotide sequence that is present inan organism, including in a virus. A naturally occurring material can bein its form as it exists in nature, and can be modified by the hand ofman such that, for example, is in an isolated form.

[0077] The term “antibody” refers to a polypeptide substantially encodedby an immunoglobulin gene or immunoglobulin genes, or antigen-bindingfragments thereof, which specifically bind and recognize an analyte(antigen). The recognized immunoglobulin genes include the kappa,lambda, alpha, gamma, delta, epsilon and mu constant region genes, aswell as the myriad immunoglobulin variable region genes. Antibodiesexist as intact immunoglobulins and as well characterizedantigen-binding fragments of an antibody, which can be produced bydigestion with a peptidase or can using recombinant DNA methods. Suchantigen-binding fragments of an antibody include, for example, Fv, Fab′and F(ab)′₂ fragments. The term “antibody,” as used herein, includesantibody fragments either produced by the modification of wholeantibodies or those synthesized de novo using recombinant DNAmethodologies. The term “immunoassay” refers to an assay that utilizesan antibody to specifically bind an analyte. An immunoassay ischaracterized by the use of specific binding properties of a particularantibody to isolate, target, and/or quantify the analyte.

[0078] The term “identical,” when used in reference to two or morepolynucleotide sequences or two or more polypeptide sequences, refers tothe residues in the sequences that are the same when aligned for maximumcorrespondence. When percentage of sequence identity is used inreference to a polypeptide, it is recognized that one or more residuepositions that are not otherwise identical can differ by a conservativeamino acid substitution, in which a first amino acid residue issubstituted for another amino acid residue having similar chemicalproperties such as a similar charge or hydrophobic or hydrophiliccharacter and, therefore, does not change the functional properties ofthe polypeptide. Where polypeptide sequences differ in conservativesubstitutions, the percent sequence identity can be adjusted upwards tocorrect for the conservative nature of the substitution. Such anadjustment can be made using well known methods, for example, scoring aconservative substitution as a partial rather than a full mismatch,thereby increasing the percentage sequence identity. Thus, for example,where an identical amino acid is given a score of 1 and anon-conservative substitution is given a score of zero, a conservativesubstitution is given a score between zero and 1. The scoring ofconservative substitutions can be calculated using any well knownalgorithm (see, for example, Meyers and Miller, Comp. Appl. Biol. Sci.4:11-17, 1988; Smith and Waterman, Adv. Appl. Math. 2:482, 1981;Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman,Proc. Natl. Acad. Sci., USA 85:2444 (1988); Higgins and Sharp, Gene73:237-244, 1988; Higgins and Sharp, CABIOS 5:151-153; 1989; Corpet etal., Nucl. Acids Res. 16:10881-10890, 1988; Huang, et al., Comp. Appl.Biol. Sci. 8:155-165, 1992; Pearson et al., Meth. Mol. Biol.,24:307-331, 1994). Alignment also can be performed by simple visualinspection and manual alignment of sequences.

[0079] The term “conservatively modified variation,” when used inreference to a particular polynucleotide sequence, refers to differentpolynucleotide sequences that encode identical or essentially identicalamino acid sequences, or where the polynucleotide does not encode anamino acid sequence, to essentially identical sequences. Because of thedegeneracy of the genetic code, a large number of functionally identicalpolynucleotides encode any given polypeptide. For instance, the codonsCGU, CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine.Thus, at every position where an arginine is specified by a codon, thecodon can be altered to any of the corresponding codons describedwithout altering the encoded polypeptide. Such nucleotide sequencevariations are “silent variations,” which can be considered a species of“conservatively modified variations.” As such, it will be recognizedthat each polynucleotide sequence disclosed herein as encoding anon-oligomerizing fluorescent protein also describes every possiblesilent variation. It will also be recognized that each codon in apolynucleotide, except AUG, which is ordinarily the only codon formethionine, and UUG, which is ordinarily the only codon for tryptophan,can be modified to yield a functionally identical molecule by standardtechniques. Accordingly, each silent variation of a polynucleotide thatdoes not change the sequence of the encoded polypeptide is implicitlydescribed herein. Furthermore, it will be recognized that individualsubstitutions, deletions or additions that alter, add or delete a singleamino acid or a small percentage of amino acids (typically less than 5%,and generally less than 1%) in an encoded sequence can be consideredconservatively modified variations, provided alteration results in thesubstitution of an amino acid with a chemically similar amino acid.Conservative amino acid substitutions providing functionally similaramino acids are well known in the art, including the following sixgroups, each of which contains amino acids that are consideredconservative substitutes for each another:

[0080] 1) Alanine (Ala, A), Serine (Ser, S), Threonine (Thr, T);

[0081] 2) Aspartic acid (Asp, D), Glutamic acid (Glu, E);

[0082] 3) Asparagine (Asn, N), Glutamine (Gln, Q);

[0083] 4) Arginine (Arg, R), Lysine (Lys, K);

[0084] 5) Isoleucine (Ile, I), Leucine (Leu, L), Methionine (Met, M),Valine (Val, V); and

[0085] 6) Phenylalanine (Phe, F), Tyrosine (Tyr, Y), Tryptophan (Trp,W).

[0086] Two or more amino acid sequences or two or more nucleotidesequences are considered to be “substantially identical” or“substantially similar” if the amino acid sequences or the nucleotidesequences share at least 80% sequence identity with each other, or witha reference sequence over a given comparison window. Thus, substantiallysimilar sequences include those having, for example, at least 85%sequence identity, at least 90% sequence identity, at least 95% sequenceidentity, or at least 99% sequence identity.

[0087] A subject nucleotide sequence is considered “substantiallycomplementary” to a reference nucleotide sequence if the complement ofthe subject nucleotide sequence is substantially identical to thereference nucleotide sequence. The term “stringent conditions” refers toa temperature and ionic conditions used in a nucleic acid hybridizationreaction. Stringent conditions are sequence dependent and are differentunder different environmental parameters. Generally, stringentconditions are selected to be about 5° C. to 20° C. lower than thethermal melting point (Tm) for the specific sequence at a defined ionicstrength and pH. The Tm is the temperature, under defined ionic strengthand pH, at which 50% of the target sequence hybridizes to a perfectlymatched probe.

[0088] The term “allelic variants” refers to polymorphic forms of a geneat a particular genetic locus, as well as cDNAs derived from mRNAtranscripts of the genes, and the polypeptides encoded by them. The term“preferred mammalian codon” refers to the subset of codons from amongthe set of codons encoding an amino acid that are most frequently usedin proteins expressed in mammalian cells as chosen from the followinglist: Gly (GGC, GGG); Glu (GAG); Asp (GAC); Val (GUG, GUC); Ala (GCC,GCU); Ser (AGC, UCC); Lys (AAG); Asn (AAC); Met (AUG); Ile (AUC); Thr(ACC); Trp (UGG); Cys (UGC); Tyr (UAU, UAC); Leu (CUG); Phe (UUC); Arg(CGC, AGG, AGA); Gln (CAG); His (CAC); and Pro (CCC).

[0089] Fluorescent molecules are useful in fluorescence resonance energytransfer, FRET, which involves a donor molecule and an acceptormolecule. To optimize the efficiency and detectability of FRET between adonor and acceptor molecule, several factors need to be balanced. Theemission spectrum of the donor should overlap as much as possible withthe excitation spectrum of the acceptor to maximize the overlapintegral. Also, the quantum yield of the donor moiety and the extinctioncoefficient of the acceptor should be as high as possible to maximizeR_(o), which represents the distance at which energy transfer efficiencyis 50%. However, the excitation spectra of the donor and acceptor shouldoverlap, as little as possible so that a wavelength region can be foundat which the donor can be excited efficiently without directly excitingthe acceptor because fluorescence arising from direct excitation of theacceptor can be difficult to distinguish from fluorescence arising fromFRET. Similarly, the emission spectra of the donor and acceptor shouldoverlap as little as possible so that the two emissions can be clearlydistinguished. High fluorescence quantum yield of the acceptor moiety isdesirable if the emission from the acceptor is to be measured either asthe sole readout or as part of an emission ratio. One factor to beconsidered in choosing the donor and acceptor pair is the efficiency offluorescence resonance energy transfer between them. Preferably, theefficiency of FRET between the donor and acceptor is at least 10%, morepreferably at least 50% and even more preferably at least 80%.

[0090] The term “fluorescent property” refers to the molar extinctioncoefficient at an appropriate excitation wavelength, the fluorescencequantum efficiency, the shape of the excitation spectrum or emissionspectrum, the excitation wavelength maximum and emission wavelengthmaximum, the ratio of excitation amplitudes at two differentwavelengths, the ratio of emission amplitudes at two differentwavelengths, the excited state lifetime, or the fluorescence anisotropy.A measurable difference in any one of these properties between wild typeAequorea GFP and a spectral variant, or a mutant thereof, is useful. Ameasurable difference can be determined by determining the amount of anyquantitative fluorescent property, e.g., the amount of fluorescence at aparticular wavelength, or the integral of fluorescence over the emissionspectrum. Determining ratios of excitation amplitude or emissionamplitude at two different wavelengths (“excitation amplitude ratioing”and “emission amplitude ratioing”, respectively) are particularlyadvantageous because the ratioing process provides an internal referenceand cancels out variations in the absolute brightness of the excitationsource, the sensitivity of the detector, and light scattering orquenching by the sample.

[0091] As used herein, the term “fluorescent protein” refers to anyprotein that can fluoresce when excited with an appropriateelectromagnetic radiation, except that chemically tagged proteins,wherein the fluorescence is due to the chemical tag, and polypeptidesthat fluoresce only due to the presence of certain amino acids such astryptophan or tyrosine, which fluoresce when exposed to ultravioletlight, are not considered fluorescent proteins for purposes of thepresent invention. In general, a fluorescent protein useful forpreparing a composition of the invention or for use in a method of theinvention is a protein that derives its fluorescence fromautocatalytically forming a chromophore. A fluorescent protein cancontain amino acid sequences that are naturally occurring or that havebeen engineered (i.e., variants or mutants). When used in reference to afluorescent protein, the term “mutant” or “variant” refers to a proteinthat is different from a reference protein. For example, a spectralvariant of Aequorea GFP can be derived from the naturally occurring GFPby engineering mutations such as amino acid substitutions into thereference GFP protein. For example ECFP is a spectral variant of GFPthat contains substitutions with respect to GFP (compare SEQ ID NOS: 2and 6).

[0092] Many cnidarians use green fluorescent proteins as energy transferacceptors in bioluminescence. The term “green fluorescent protein” isused broadly herein to refer to a protein that fluoresces green light,for example, Aequorea GFP (SEQ ID NO: 2). GFPs have been isolated fromthe Pacific Northwest jellyfish, Aequorea victoria, the sea pansy,Renilla reniformis, and Phialidium gregarium (Ward et al., Photochem.Photobiol. 35:803-808, 1982; Levine et al., Comp. Biochem. Physiol.72B:77-85, 1982, each of which is incorporated herein by reference).Similarly, reference is made herein to “red fluorescent proteins”, whichfluoresce red, “cyan fluorescent proteins,” which fluoresce cyan, andthe like. RFPs, for example, have been isolated from the coral,Discosoma (Matz et al., supra, 1999).

[0093] A variety of Aequorea GFP-related fluorescent proteins havinguseful excitation and emission spectra have been engineered by modifyingthe amino acid sequence of a naturally occurring GFP from A. victoria(see Prasher et al., Gene 111:229-233, 1992; Heim et al., Proc. Natl.Acad. Sci., USA 91:12501-12504, 1994; U.S. Ser. No. 08/337,915, filedNov. 10, 1994; International application PCT/US95/14692, each of whichis incorporated herein by reference). As used herein, reference to a“related fluorescent protein” refers to a fluorescent protein that has asubstantially identical amino acid sequence when compared to a referencefluorescent protein. In general, a related fluorescent protein, whencompared to the reference fluorescent protein sequence, has a contiguoussequence of at least about 150 amino acids that shares at least about85% sequence identity with the reference fluorescent protein, andparticularly has a contiguous sequence of at least about 200 amino acidsthat shares at least about 95% sequence identity with the referencefluorescent protein. Thus, reference is made herein to an“Aequorea-related fluorescent protein” or to a “GFP-related fluorescentprotein,” which is exemplified by the various spectral variants and GFPmutants that have amino acid sequences that are substantially identicalto A. victoria GFP (SEQ ID NO: 2), to a “Discosoma-related fluorescentprotein” or a “DsRed-related fluorescent related protein,” which isexemplified by the various mutants that have amino acid sequencessubstantially identical to that of DsRed (SEQ ID NO: 12), and the like,for example, a Renilla-related fluorescent protein or aPhialidium-related fluorescent protein.

[0094] The term “mutant” or “variant” also is used herein in referenceto a fluorescent protein to refer to a fluorescent protein that containsa mutation with respect to a corresponding wild type fluorescentprotein. In addition, reference is made herein the a “spectral variant”or “spectral mutant” of a fluorescent protein to indicate a mutantfluorescent protein that has a different fluorescence characteristicwith respect to the corresponding wild type fluorescent protein. Forexample, CFP, YFP, ECFP (SEQ ID NO: 6), EYFP-V68L/Q69K (SEQ ID NO: 10),and the like are GFP spectral variants.

[0095] Aequorea GFP-related fluorescent proteins include, for example,wild type (native) Aequorea victoria GFP (Prasher et al., supra, 1992;see, also, SEQ ID NO: 2), allelic variants of SEQ ID NO: 2, for example,a variant having a Q80R substitution (Chalfie et al., Science263:802-805, 1994, which is incorporated herein by reference); andspectral variants of GFP such as CFP, YFP, and enhanced and otherwisemodified forms thereof (U.S. Pat. Nos. 6,150,176; 6,124,128; 6,077,707;6,066,476; 5,998,204; and 5,777,079, each of which is incorporatedherein by reference), including GFP-related fluorescent proteins havingone or more folding mutations, and fragments of the proteins that arefluorescent, for example, an A. victoria GFP from which the twoN-terminal amino acid residues have been removed. Several of thesefluorescent proteins contain different aromatic amino acids within thecentral chromophore and fluoresce at a distinctly shorter wavelengththan the wild type GFP species. For example, the engineered GFP proteinsdesignated P4 and P4-3 contain, in addition to other mutations, thesubstitution Y66H; and the engineered GFP proteins designated W2 and W7contain, in addition to other mutations, Y66W.

[0096] Folding mutations in Aequorea GFP-related fluorescent proteinsimprove the ability of the fluorescent proteins to fold at highertemperatures, and to be more fluorescent when expressed in mammaliancells, but have little or no effect on the peak wavelengths ofexcitation and emission. If desired, these mutations can be combinedwith additional mutations that influence the spectral properties of GFPto produce proteins with altered spectral and folding properties, and,particularly, with mutations that reduce or eliminate the propensity ofthe fluorescent proteins to oligomerize. Folding mutations, with respectto SEQ ID NO: 2, include the substitutions F64L, V68L, S72A, T44A, F99S,Y145F, N146I, M153T, M153A, V163A, I167T, S175G, S205T, and N212K.

[0097] The term “loop domain” refers to an amino acid sequence of anAequorea-related fluorescent protein that connects the amino acidsinvolved in the secondary structure of the eleven strands of theθ-barrel or the central 1-helix (residues 56-72). The term “fluorescentprotein moiety,” when used in reference to a fluorescent protein, refersto a portion of the amino acid sequence of the fluorescent protein that,when the amino acid sequence of the fluorescent protein substrate isoptimally aligned with the amino acid sequence of a naturally occurringfluorescent protein, lies between the amino terminal and carboxyterminal amino acids, inclusive, of the amino acid sequence of thenaturally occurring fluorescent protein, and comprises a chromophore,which fluoresces upon exposure to an appropriate wavelength of light.

[0098] Fluorescent proteins fused to target proteins can be preparedusing recombinant DNA methods, and used as markers to identify thelocation and amount of the target protein produced. Accordingly, thepresent invention provides fusion proteins comprising anon-oligomerizing fluorescent protein moiety and a polypeptide ofinterest. The polypeptide of interest can be of any length, for example,about 15 amino acid residues, about 50 residues, about 150 residues, orup to about 1000 amino acid residues or more, provided that thefluorescent protein component of the fusion protein can fluoresce or canbe induced to fluoresce when exposed to electromagnetic radiation of theappropriate wavelength. The polypeptide of interest can be, for example,a peptide tag such as a polyhistidine sequence, a c-myc epitope, a FLAGepitope, and the like; can be an enzyme, which can be used to effect afunction in a cell expressing a fusion protein comprising the enzyme orto identify a cell containing the fusion protein; can be a protein to beexamined for an ability to interact with one or more other proteins in acell, or any other protein as disclosed herein or otherwise desired.

[0099] As disclosed herein, the Discosoma (coral) red fluorescentprotein, DsRed, can be used as a complement to or alternative for a GFPor spectral variant thereof. Amino acid residues of DsRed thatcorrespond to those of GFP have been identified, and mutations ofselected amino acid residues, based on knowledge of the correspondingstructures, has allowed the identification of DsRed mutants havingdifferent fluorescent properties as compared to wild type DsRed (seeExample 2). In addition, DsRed is shown to have a propensity tooligomerize, similar to that dimerization that occurs for GFPs. As such,mutations can be made in DsRed and the identified mutants thatcorrespond to those introduced into GFP that reduce or eliminatedimerization of GFPs (Examples 1 and 3). Furthermore, X-raycrystallography of DsRed and computer processing can be used to confirmthat the optimal amino acid residues have been selected for mutation toreduce or eliminate oligomerization, similar to the model of the crystalstructure of Aequorea GFP that was prepared (see U.S. Pat. No.6,124,128). As further disclosed herein, non-oligomerizing tandem DsRedfluorescent proteins can be constructed, and the strategy used to designthe tandem DsRed proteins can be applied to other fluorescent proteins(see Example 4).

[0100] Fluorescent characteristics of Aequorea GFP-related fluorescentproteins depend, in part, on the electronic environment of thechromophore. In general, amino acids that are within about 0.5 nm of thechromophore influence the electronic environment of the chromophore.Therefore, substitution of such amino acids can produce fluorescentproteins with altered fluorescent characteristics. In the excited state,electron density tends to shift from the phenolate towards the carbonylend of the chromophore. Therefore, placement of increasing positivecharge near the carbonyl end of the chromophore tends to decrease theenergy of the excited state and cause a red-shift in the absorbance andemission wavelength maximum of the protein. Decreasing a positive chargenear the carbonyl end of the chromophore tends to have the oppositeeffect, causing a blue-shift in the protein's wavelengths. Similarly,mutations have been introduced into DsRed to produce mutants havingaltered fluorescence characteristics (see Example 2).

[0101] Amino acids with charged (ionized D, E, K, and R), dipolar (H, N,Q, S, T, and uncharged D, E and K), and polarizable side groups (e.g.,C, F, H, M, W and Y) are useful for altering the ability of fluorescentproteins to oligomerize, especially when they substitute an amino acidwith an uncharged, nonpolar or non-polarizable side chain (see Examples1 and 3). As disclosed herein, substitution of hydrophobic residues thatwere predicted to be involved in self-association of GFP withpositively-charged residues reduced or eliminated dimerization. However,other non-conservative amino acid substitutions also can be introducedsimilarly or at neighboring positions in the interacting regions of theproteins, thus disrupting the localized structure of the protein,provided the substitutions do not undesirably affect the fluorescentproperties of the proteins. Accordingly, the present invention providesnon-oligomerizing fluorescent proteins.

[0102] A fusion protein, which includes a non-oligomerizing fluorescentprotein, for example, a non-oligomerizing tandem fluorescent protein,operatively linked to one or more polypeptides of interest also isprovided. The polypeptides of the fusion protein can be linked throughpeptide bonds, or the non-oligomerizing fluorescent protein can belinked to the polypeptide of interest through a linker molecule. In oneembodiment, the fusion protein is expressed from a recombinant nucleicacid molecule containing a polynucleotide encoding a non-oligomerizingfluorescent protein operatively linked to one or more polynucleotidesencoding one or more polypeptides of interest.

[0103] A polypeptide of interest can be any polypeptide, including, forexample, a peptide tag such as a polyhistidine peptide, or a cellularpolypeptide such as an enzyme, a G-protein, a growth factor receptor, ora transcription factor; and can be one of two or more proteins that canassociate to form a complex. In one embodiment, the fusion protein is atandem non-oligomerizing fluorescent protein construct, which includes adonor non-oligomerizing fluorescent protein, an acceptornon-oligomerizing fluorescent protein, and a peptide linker moietycoupling said donor and said acceptor, wherein cyclized amino acids ofthe donor emit light characteristic of said donor, and wherein the donorand the acceptor exhibit fluorescence resonance energy transfer when thedonor is excited, and the linker moiety does not substantially emitlight to excite the donor. As such, a fusion protein of the inventioncan include two or more operatively linked non-oligomerizing fluorescentproteins, which can be linked directly or indirectly, and can furthercomprise one or more polypeptides of interest.

[0104] A tandem non-oligomerizing fluorescent protein includes a donor,comprising a first fluorescent protein, an acceptor, comprising a secondfluorescent protein, and a peptide linker moiety operatively linking thedonor and the acceptor. In such a tandem non-oligomerizing fluorescentprotein, the first fluorescent protein and second fluorescent proteinare different, and at least the first fluorescent protein or the secondfluorescent protein is a non-oligomerizing tandem fluorescent protein ofthe invention. In addition, the cyclized amino acids of the donor emitlight characteristic of the donor, and the donor and the acceptorexhibit fluorescence resonance energy transfer when the donor isexcited, and the linker moiety does not substantially emit light toexcite the acceptor.

[0105] It should be recognized the reference to a “first” fluorescentprotein or a “second” fluorescent protein or the like is used only toconveniently refer to a particular protein, but is not intended toindicate any order or importance of the protein. As such, wherereference is made, for example, to a first non-oligomerizing tandemfluorescent protein and a second non-oligomerizing fluorescent protein,it will be recognized that either of the first or second fluorescentproteins can be the non-oligomerizing tandem fluorescent protein or canbe the non-oligomerizing fluorescent protein.

[0106] In one embodiment, each of the first fluorescent protein and thesecond fluorescent protein is a non-oligomerizing tandem fluorescentprotein in a tandem non-oligomerizing fluorescent protein of theinvention. For example, the non-oligomerizing tandem fluorescent proteincan comprise two or more Discosoma RFPs or a fluorescent protein relatedto a Discosoma RFP, such as a DsRed protein having an amino acidsequence as set forth in SEQ ID NO: 12 or a mutant DsRed protein such asSEQ ID NO:12 containing an I125R mutation. In another embodiment, thefirst fluorescent protein is a non-oligomerizing tandem fluorescentprotein, and the second fluorescent protein is a non-oligomerizingfluorescent protein. The non-oligomerizing fluorescent protein cancontain a mutation of an amino acid residue corresponding to A206, L221,F223, or a combination thereof of SEQ ID NO:2, for example, a mutationcorresponding to S65G/S72A/T203Y/H231L in SEQ ID NO:2; a mutationcorresponding to

[0107] The present invention also provides a polynucleotide encoding anon-oligomerizing fluorescent protein, which can be a non-oligomerizingtandem fluorescent protein, as well as to a vector containing such apolynucleotide, and a host cell containing a polynucleotide or vector.Also provided is a recombinant nucleic acid molecule, which includes atleast one polynucleotide encoding a non-oligomerizing fluorescentprotein operatively linked to one or more other polynucleotides. The oneor more other polynucleotides can be, for example, a transcriptionregulatory element such as a promoter or polyadenylation signalsequence, or a translation regulatory element such as a ribosome bindingsite. Such a recombinant nucleic acid molecule can be contained in avector, which can be an expression vector, and the nucleic acid moleculeor the vector can be contained in a host cell.

[0108] The vector generally contains elements required for replicationin a prokaryotic or eukaryotic host system or both, as desired. Suchvectors, which include plasmid vectors and viral vectors such asbacteriophage, baculovirus, retrovirus, lentivirus, adenovirus, vacciniavirus, semliki forest virus and adeno-associated virus vectors, are wellknown and can be purchased from a commercial source (Promega, MadisonWis.; Stratagene, La Jolla Calif.; GIBCO/BRL, Gaithersburg Md.) or canbe constructed by one skilled in the art (see, for example, Meth.Enzymol., Vol. 185, Goeddel, ed. (Academic Press, Inc., 1990); Jolly,Canc. Gene Ther. 1:51-64, 1994; Flotte, J. Bioenerg. Biomemb. 25:37-42,1993; Kirshenbaum et al., J. Clin. Invest. 92:381-387, 1993; each ofwhich is incorporated herein by reference).

[0109] A vector for containing a polynucleotide encoding anon-oligomerizing fluorescent protein can be a cloning vector or anexpression vector, and can be a plasmid vector, viral vector, and thelike. Generally, the vector contains a selectable marker independent ofthat encoded by a polynucleotide of the invention, and further cancontain transcription or translation regulatory elements, including apromoter sequence, which can provide tissue specific expression of apolynucleotide operatively linked thereto, which can, but need not, bethe polynucleotide encoding the non-oligomerizing fluorescent protein,for example, a non-oligomerizing tandem fluorescent protein, thusproviding a means to select a particular cell type from among a mixedpopulation of cells containing the introduced vector and recombinantnucleic acid molecule contained therein.

[0110] Where the vector is a viral vector, it can be selected based onits ability to infect one or few specific cell types with relativelyhigh efficiency. For example, the viral vector also can be derived froma virus that infects particular cells of an organism of interest, forexample, vertebrate host cells such as mammalian host cells. Viralvectors have been developed for use in particular host systems,particularly mammalian systems and include, for example, retroviralvectors, other lentivirus vectors such as those based on the humanimmunodeficiency virus (HIV), adenovirus vectors, adeno-associated virusvectors, herpesvirus vectors, vaccinia virus vectors, and the like (seeMiller and Rosman, BioTechniques 7:980-990, 1992; Anderson et al.,Nature 392:25-30 Suppl., 1998; Verma and Somia, Nature 389:239-242,1997; Wilson, New Engl. J. Med. 334:1185-1187 (1996), each of which isincorporated herein by reference).

[0111] Recombinant production of a non-oligomerizing fluorescentprotein, which can be a component of a fusion protein, involvesexpressing a polypeptide encoded by a polynucleotide. A polynucleotideencoding the non-oligomerizing fluorescent protein is a useful startingmaterials. Polynucleotides encoding fluorescent protein are disclosedherein or otherwise known in the art, and can be obtained using routinemethods, then can be modified such that the encoded fluorescent proteinlacks a propensity to oligomerize. For example, a polynucleotideencoding a GFP can be isolated by PCR of cDNA from A. victoria usingprimers based on the DNA sequence of Aequorea GFP (SEQ ID NO: 2). PCRmethods are well known and routine in the art (see, for example, U.S.Pat. No. 4,683,195; Mullis et al., Cold Spring Harbor Symp. Quant. Biol.51:263, 1987; Erlich, ed., “PCR Technology” (Stockton Press, NY, 1989)).A non-oligomerizing form of the fluorescent protein then can be made bysite-specific mutagenesis of the polynucleotide encoding the fluorescentprotein, or by random mutagenesis caused by increasing the error rate ofPCR of the original polynucleotide with 0.1 mM MnCl₂ and unbalancednucleotide concentrations (Example, 1; see, also, U.S. Pat. No.6,066,476). Similarly, a non-oligomerizing tandem fluorescent proteincan be expressed from a polynucleotide prepared by PCR using primersthat can encode, for example, a peptide linker, which operatively linksa first monomer and at least a second monomer of a fluorescent protein(see Example 4).

[0112] The construction of expression vectors and the expression of apolynucleotide in transfected cells involves the use of molecularcloning techniques also well known in the art (see Sambrook et al., In“Molecular Cloning: A Laboratory Manual” (Cold Spring Harbor LaboratoryPress 1989); “Current Protocols in Molecular Biology” (eds., Ausubel etal.; Greene Publishing Associates, Inc., and John Wiley & Sons, Inc.1990 and supplements). Expression vectors contain expression controlsequences operatively linked to a polynucleotide sequence of interest,for example, that encoding a non-oligomerizing fluorescent protein, asindicated above. The expression vector can be adapted for function inprokaryotes or eukaryotes by inclusion of appropriate promoters,replication sequences, markers, and the like. An expression vector canbe transfected into a recombinant host cell for expression of anon-oligomerizing fluorescent protein, and host cells can be selected,for example, for high levels of expression in order to obtain a largeamount of isolated protein. A host cell can be maintained in cellculture, or can be a cell in vivo in an organism. A non-oligomerizingfluorescent protein can be produced by expression from a polynucleotideencoding the protein in a host cell such as E. coli. AequoreaGFP-related fluorescent proteins, for example, are best expressed bycells cultured between about 15° C. and 30° C., although highertemperatures such as 37° C. can be used. After synthesis, thefluorescent proteins are stable at higher temperatures and can be usedin assays at such temperatures.

[0113] An expressed non-oligomerizing fluorescent protein, which can bea non-oligomerizing tandem fluorescent protein and can be operativelylinked to a first polypeptide of interest, further can be linked to asecond polypeptide of interest, for example, a peptide tag, which can beused to facilitate isolation of the non-oligomerizing fluorescentprotein, including any other polypeptides linked thereto. For example, apolyhistidine tag containing, for example, six histidine residues, canbe incorporated at the N-terminus or C-terminus of the non-oligomerizingfluorescent protein, which then can be isolated in a single step usingnickel-chelate chromatography (see Example, 1). Additional peptide tags,including a c-myc peptide, a FLAG epitope, or any ligand (or cognatereceptor), including any peptide epitope (or antibody, or antigenbinding fragment thereof, that specifically binds the epitope are wellknown in the art and similarly can be used. (see, for example, Hopp etal., Biotechnology 6:1204 (1988); U.S. Pat. No. 5,011,912, each of whichis incorporated herein by reference).

[0114] Kits also are provided to facilitate and, where desired,standardize the compositions of the invention and the uses thereof. Akit can contain one or more compositions of the invention, for example,one or a plurality of non-oligomerizing fluorescent proteins, which canbe a portion of a fusion protein, or one or a plurality ofpolynucleotides that encode the polypeptides. The non-oligomerizingfluorescent protein can be a mutated fluorescent protein having areduced propensity to oligomerize or can be a non-oligomerizing tandemfluorescent protein and, where the kit comprises a plurality ofnon-oligomerizing fluorescent proteins, the plurality can be a pluralityof the mutated non-oligomerizing fluorescent proteins, or of thenon-oligomerizing tandem fluorescent proteins, or a combination thereof.

[0115] A kit of the invention also can contain one or a plurality ofrecombinant nucleic acid molecules, which encode, in part,non-oligomerizing fluorescent proteins, which can be the same ordifferent, and can further include, for example, an operatively linkedsecond polynucleotide containing or encoding a restriction endonucleaserecognition site or a recombinase recognition site, or any polypeptideof interest. In addition, the kit can contain instructions for using thecomponents of the kit, particularly the compositions of the inventionthat are contained in the kit.

[0116] Such kits can be particularly useful where they provide aplurality of different non-oligomerizing fluorescent proteins becausethe artisan can conveniently select one or more proteins having thefluorescent properties desired for a particular application. Similarly,a kit containing a plurality of polynucleotides encoding differentnon-oligomerizing fluorescent proteins provides numerous advantages. Forexample, the polynucleotides can be engineered to contain convenientrestriction endonuclease or recombinase recognition sites, thusfacilitating operative linkage of the polynucleotide to a regulatoryelement or to a polynucleotide encoding a polypeptide of interest or, ifdesired, for operatively linking two or more the polynucleotidesencoding the non-oligomerizing fluorescent proteins to each other.

[0117] A non-oligomerizing fluorescent protein of the invention isuseful in any method that employs a fluorescent proteins. Thus, thenon-oligomerizing fluorescent proteins, including the non-oligomerizingtandem fluorescent proteins, are useful as fluorescent markers in themany ways fluorescent markers already are used, including, for example,coupling non-oligomerizing fluorescent proteins to antibodies,polynucleotides or other receptors for use in detection assays such asimmunoassays or hybridization assays, or to track the movement ofproteins in cells. For intracellular tracking studies, a first (orother) polynucleotide encoding the non-oligomerizing fluorescent proteinis fused to a second (or other) polynucleotide encoding a protein ofinterest and the construct, if desired, can be inserted into anexpression vector. Upon expression inside the cell, the protein ofinterest can be localized based on fluorescence, without concern thatlocalization of the protein is an artifact caused by oligomerization ofthe fluorescent protein component of the fusion protein. In oneembodiment of this method, two proteins of interest independently arefused with two non-oligomerizing fluorescent proteins that havedifferent fluorescent characteristics.

[0118] The non-oligomerizing fluorescent proteins of this invention areuseful in systems to detect induction of transcription. For example, anucleotide sequence encoding a non-oligomerizing tandem fluorescentprotein can be fused to a promoter or other expression control sequenceof interest, which can be contained in an expression vector, theconstruct can be transfected into a cell, and induction of the promoter(or other regulatory element) can be measured by detecting the presenceor amount of fluorescence, thereby allowing a means to observe theresponsiveness of a signaling pathway from receptor to promoter.

[0119] A non-oligomerizing fluorescent protein of the invention also isuseful in applications involving FRET, which can detect events as afunction of the movement of fluorescent donors and acceptors towards oraway from each other. One or both of the donor/acceptor pair can be anon-oligomerizing fluorescent protein, for example, a donor GFP having aT203I mutation and an acceptor GFP having the mutation T203X, wherein Xis an aromatic amino acid, for example, T203Y, T203W, or T203H (see U.S.Pat. Nos. 6,124,128 and 6,066,476). Another useful donor/acceptor pairincludes a donor having the mutations S72A, K79R, Y145F, M153A and T203I(with a excitation peak of 395 nm and an emission peak of 511 nm) and anacceptor having the mutations S65G, S72A, K79R, and T203Y. Such adonor/acceptor pair provides a wide separation between the excitationand emission peaks of the donor, and provides good overlap between thedonor emission spectrum and the acceptor excitation spectrum. Othernon-oligomerizing red fluorescent proteins or red-shifted mutants asdisclosed herein can also be used as the acceptor in such a pair.

[0120] FRET can be used to detect cleavage of a substrate having thedonor and acceptor coupled to the substrate on opposite sides of thecleavage site. Upon cleavage of the substrate, the donor/acceptor pairphysically separate, eliminating FRET. Such an assay can be performed,for example, by contacting the substrate with a sample, and determininga qualitative or quantitative change in FRET (see, for example, U.S.Pat. No. 5,741,657, which is incorporated herein by reference). Anon-oligomerizing fluorescent protein donor/acceptor pair also can bepart of a fusion protein coupled by a peptide having a proteolyticcleavage site (see, for example, U.S. Pat. No. 5,981,200, which isincorporated herein by reference). FRET also can be used to detectchanges in potential across a membrane. For example, a donor andacceptor can be placed on opposite sides of a membrane such that onetranslates across the membrane in response to a voltage change, therebyproducing a measurable FRET (see, for example, U.S. Pat. No. 5,661,035,which is incorporated herein by reference).

[0121] A non-oligomerizing fluorescent protein of the invention isuseful for making a fluorescent substrate for a protein kinase. Such asubstrate incorporates an amino acid sequence recognizable by a proteinkinases and, upon phosphorylation, the non-oligomerizing fluorescentprotein undergoes a change in a fluorescent property. Such substratesare useful for detecting and measuring protein kinase activity in asample of a cell, upon transfection and expression of the substrate.Preferably, the kinase recognition site is placed within about 20 aminoacids of a terminus of the non-oligomerizing fluorescent protein, or ina loop domain of the protein (see U.S. Ser. No. 08/680,877, filed Jul.16, 1996, which is incorporated herein by reference). Similarly, aprotease recognition site also can be introduced into a loop domain suchthat, upon cleavage, the fluorescent property changes in a measurablefashion.

[0122] Fluorescence in a sample generally is measured using afluorimeter, wherein excitation radiation from an excitation sourcehaving a first wavelength, passes through excitation optics, which causethe excitation radiation to excite the sample. In response, anon-oligomerizing fluorescent protein in the sample emits radiationhaving a wavelength that is different from the excitation wavelength.Collection optics then collect the emission from the sample. The devicecan include a temperature controller to maintain the sample at aspecific temperature while it is being scanned, and can have amulti-axis translation stage, which moves a microtiter plate holding aplurality of samples in order to position different wells to be exposed.The multi-axis translation stage, temperature controller, auto-focusingfeature, and electronics associated with imaging and data collection canbe managed by an appropriately programmed digital computer, which alsocan transform the data collected during the assay into another formatfor presentation. This process can be miniaturized and automated toenable screening many thousands of compounds in a high throughputformat. These and other methods of performing assays on fluorescentmaterials are well known in the art (see, for example, Lakowicz,“Principles of Fluorescence Spectroscopy” (Plenum Press 1983); Herman,“Resonance energy transfer microscopy” In “Fluorescence Microscopy ofLiving Cells in Culture” Part B, Meth. Cell Biol. 30:219-243 (ed. Taylorand Wang; Academic Press 1989); Turro, “Modem Molecular Photochemistry”(Benjamin/Cummings Publ. Co., Inc. 1978), pp. 296-361, each of which isincorporated herein by reference).

[0123] Accordingly, the present invention provides a method foridentifying the presence of a molecule in a sample. Such a method can beperformed, for example, by linking a non-oligomerizing fluorescentprotein of the invention to the molecule, and detecting fluorescence dueto the non-oligomerizing fluorescent protein in a sample suspected ofcontaining the molecule. The molecule to be detected can be apolypeptide, a polynucleotide, or any other molecule, including, forexample, an antibody, an enzyme, or a receptor, and thenon-oligomerizing fluorescent protein can be a non-oligomerizing tandemfluorescent protein.

[0124] The sample to be examined can be any sample, including abiological sample, an environmental sample, or any other sample forwhich it is desired to determine whether a particular molecule ispresent therein. Preferably, the sample includes a cell or an extractthereof. The cell can be obtained from a vertebrate, including a mammalsuch as a human, or from an invertebrate, and can be a cell from a plantor an animal. The cell can be obtained from a culture of such cells, forexample, a cell line, or can be isolated from an organism. As such, thecell can be contained in a tissue sample, which can be obtained from anorganism by any means commonly used to obtain a tissue sample, forexample, by biopsy of a human. Where the method is performed using anintact living cell or a freshly isolated tissue or organ sample, thepresence of a molecule of interest in living cells can be identified,thus providing a means to determine, for example, the intracellularcompartmentalization of the molecule. The use of the non-oligomerizingfluorescent proteins of the invention for such a purpose provides asubstantial advantage in that the likelihood of aberrant identificationor localization due to oligomerization the fluorescent protein isgreatly minimized.

[0125] A non-oligomerizing fluorescent protein can be linked to themolecule directly or indirectly, using any linkage that is stable underthe conditions to which the protein-molecule complex is to be exposed.Thus, the fluorescent protein and molecule can be linked via a chemicalreaction between reactive groups present on the protein and molecule, orthe linkage can be mediated by linker moiety, which contains reactivegroups specific for the fluorescent protein and the molecule. It will berecognized that the appropriate conditions for linking thenon-oligomerizing fluorescent protein and the molecule are selecteddepending, for example, on the chemical nature of the molecule and thetype of linkage desired. Where the molecule of interest is apolypeptide, a convenient means for linking a non-oligomerizingfluorescent protein and the molecule is by expressing them as a fusionprotein from a recombinant nucleic acid molecule, which comprises apolynucleotide encoding, for example, a non-oligomerizing tandemfluorescent protein operatively linked to a polynucleotide encoding thepolypeptide molecule.

[0126] A method of identifying an agent or condition that regulates theactivity of an expression control sequence also is provided. Such amethod can be performed, for example, by exposing a recombinant nucleicacid molecule, which includes a polynucleotide encoding anon-oligomerizing fluorescent protein operatively linked to anexpression control sequence, to an agent or condition suspected of beingable to regulate expression of a polynucleotide from the expressioncontrol sequence, and detecting fluorescence of the non-oligomerizingfluorescent protein due to such exposure. Such a method is useful, forexample, for identifying chemical or biological agents, includingcellular proteins, that can regulate expression from the expressioncontrol sequence, including cellular factors involved in the tissuespecific expression from the regulatory element. As such, the expressioncontrol sequence can be a transcription regulatory element such as apromoter, enhancer, silencer, intron splicing recognition site,polyadenylation site, or the like; or a translation regulatory elementsuch as a ribosome binding site.

[0127] The non-oligomerizing fluorescent proteins of the invention alsoare useful in a method of identifying a specific interaction of a firstmolecule and a second molecule. Such a method can be performed, forexample, by contacting the first molecule, which is linked to a donorfirst non-oligomerizing fluorescent protein, and the second molecule,which is linked to an acceptor second non-oligomerizing fluorescentprotein, under conditions that allow a specific interaction of the firstmolecule and second molecule; exciting the donor; and detectingfluorescence resonance energy transfer from the donor to the acceptor,thereby identifying a specific interaction of the first molecule and thesecond molecule. The conditions for such an interaction can be anyconditions under which is expected or suspected that the molecules canspecifically interact. In particular, where the molecules to be examinedare cellular molecules, the conditions generally are physiologicalconditions. As such, the method can be performed in vitro usingconditions of buffer, pH, ionic strength, and the like, that mimicphysiological conditions, or the method can be performed in a cell orusing a cell extract.

[0128] The first and second molecules can be cellular proteins that arebeing investigated to determine whether the proteins specificallyinteract, or to confirm such an interaction. Such first and secondcellular proteins can be the same, where they are being examined, forexample, for an ability to oligomerize, or they can be different wherethe proteins are being examined as specific binding partners involved,for example, in an intracellular pathway. The first and second moleculesalso can be a polynucleotide and a polypeptide, for example, apolynucleotide known or to be examined for transcription regulatoryelement activity and a polypeptide known or being tested fortranscription factor activity. For example, the first molecule cancomprise a plurality of nucleotide sequences, which can be random or canbe variants of a known sequence, that are to be tested for transcriptionregulatory element activity, and the second molecule can be atranscription factor, such a method being useful for identifying noveltranscription regulatory elements having desirable activities.

[0129] The present invention also provides a method for determiningwhether a sample contains an enzyme. Such a method can be performed, forexample, by contacting a sample with a tandem non-oligomerizingfluorescent protein of the invention; exciting the donor, anddetermining a fluorescence property in the sample, wherein the presenceof an enzyme in the sample results in a change in the degree offluorescence resonance energy transfer. Similarly, the present inventionrelates to a method for determining the activity of an enzyme in a cell.Such a method can be performed, for example, providing a cell thatexpresses a tandem non-oligomerizing fluorescent protein construct,wherein the peptide linker moiety comprises a cleavage recognition aminoacid sequence specific for the enzyme coupling the donor and theacceptor; exciting said donor, and determining the degree offluorescence resonance energy transfer in the cell, wherein the presenceof enzyme activity in the cell results in a change in the degree offluorescence resonance energy transfer.

[0130] Also provided is a method for determining the pH of a sample.Such a method can be performed, for example, by contacting the samplewith a first non-oligomerizing fluorescent protein, which can be anon-oligomerizing tandem fluorescent protein, wherein the emissionintensity of the first non-oligomerizing fluorescent protein changes aspH varies between pH 5 and pH 10; exciting the indicator; anddetermining the intensity of light emitted by the firstnon-oligomerizing fluorescent protein at a first wavelength, wherein theemission intensity of the first non-oligomerizing fluorescent proteinindicates the pH of the sample. The first non-oligomerizing fluorescentprotein useful in this method, or in any method of the invention, cancomprise two DsRed monomers as set forth in SEQ ID NO:12, or a mutantthereof such as an I125R mutant, operatively linked, for example, by apeptide having an amino acid sequence of SEQ ID NO:26; or can have anamino acid sequence of SEQ ID NO: 2, or a sequence substantiallyidentical thereto, for example, having the mutationsS65G/S72A/T203Y/H231L with respect to SEQ ID NO:2, or the mutationsS65G/V68L/Q69K/S72A/T203Y/H23 IL with respect to SEQ ID NO:2; or themutations K26R/F64L/S65T/Y66W/N1461/M153T/V163A/N164H/H231L with respectto SEQ ID NO:2; or any of the above mutated non-oligomerizingfluorescent protein further having a mutation corresponding to H148G orH148Q with respect to SEQ ID NO: 2. It will be recognized that suchnon-oligomerizing fluorescent proteins similarly are useful, eitheralone or in combination, for the variously disclosed methods of theinvention.

[0131] The sample used in a method for determining the pH of a samplecan be any sample, including, for example, a biological tissue sample,or a cell or a fraction thereof. In addition, the method can furtherinclude contacting the sample with a second non-oligomerizingfluorescent protein, wherein the emission intensity of the secondnon-oligomerizing fluorescent protein changes as pH varies from 5 to 10,and wherein the second non-oligomerizing fluorescent protein emits at asecond wavelength that is distinct from the first wavelength; excitingthe second non-oligomerizing fluorescent protein; determining theintensity of light emitted by the second non-oligomerizing fluorescentprotein at the second wavelength; and comparing the fluorescence at thesecond wavelength to the fluorescence at the first wavelength. The first(or second) non-oligomerizing fluorescent protein can include atargeting sequence, for example, a cell compartmentalization domain sucha domain that targets the non-oligomerizing fluorescent protein in acell to the cytosol, the endoplasmic reticulum, the mitochondrialmatrix, the chloroplast lumen, the medial trans-Golgi cistemae, a lumenof a lysosome, or a lumen of an endosome. For example, the cellcompartmentalization domain can include amino acid residues 1 to 81 ofhuman type II membrane-anchored protein galactosyltransferase, or aminoacid residues 1 to 12 of the presequence of subunit IV of cytochrome coxidase.

[0132] The following examples are intended to illustrate, but not limit,the present invention.

EXAMPLE 1 Preparation and characterization of Non-OligomerizingFluorescent Proteins

[0133] This example demonstrates that mutations can be introduced intoGFP spectral variants that reduce or eliminate the ability of theproteins to oligomerize.

[0134] ECFP (SEQ ID NO: 6) and EYFP-V68L/Q69K (SEQ ID NO: 10) at thedimer interface were subcloned into the bacterial expression vectorPRSET_(B) (Invitrogen Corp., La Jolla Calif.), creating an N-terminalHis₆ tag on the of ECFP (SEQ ID NO: 6) and EYFP-V68L/Q69K (SEQ ID NO:10), which allowed purification of the bacterially expressed proteins ona nickel-agarose (Qiagen) affinity column. All dimer-related mutationsin the cDNAs were created by site-directed mutagenesis using theQuickChange mutagenesis kit (Stratagene), then expressed and purified inthe same manner. All cDNAs were sequenced to ensure that only thedesired mutations existed.

[0135] EYFP-V68L/Q69K (SEQ ID NO: 10) was mutagenized using theQuickChange kit (Stratagene). The overlapping mutagenic primers weredesignated “top” for the 5′ primer and “bottom” for the 3′ primer andare designated according to the particular mutation introduced (seeTable 1). All primers had a melting temperature greater than 70° C. Themutations were made as close to the center of the primers as possibleand all primers were purified by polyacrylamide gel electrophoresis. Theprimers are shown in a 5′ to 3′ orientation, with mutagenized codonsunderlined (Table 1). TABLE 1 A206K top CAG TCC AAG CTG AGC AAA GAC CCCAAC GAG AAG (SEQ ID NO:13) CGC GAT CAC A206K bottom GTG ATC GCG CTT CTCGTT GGG GTC TTT GCT CAG (SEQ ID NO:14) CTT GGA CTG L221K top CAC ATG GTCCTG AAG GAG TTC GTG ACC GCC GCC (SEQ ID NO:15) GGG L221K bottom CCC GGCGGC GGT CAC GAA CTC CTT CAG GAC CAT (SEQ ID NO:16) GTG F223R top CAC ATGGTC CTG CTG GAG CGC GTG ACC GCC GCC (SEQ ID NO:17) GGG F223R bottom CCCGGC GGC GGT CAC GCG CTC CAG CAG GAC CAT (SEQ ID NO:18) GTG L221K/F223RCAC ATC GTC CTG AAGGAG CGC GTG ACC GCC GCC (SEQ ID NO:19) top GGGL221K/F223R CCC GGC GGC GGT CAC GCG CTC CTT CAG GAC CAT (SEQ ID NO:20)bot. GTG

[0136] For protein expression, plasmids containing cDNAs for the variousEYFP-V68L/Q69K (SEQ ID NO: 10) mutants were transformed into E. colistrain JM109 and grown to an OD₆₀₀ of 0.6 in LB containing 100 μg/mlampicillin at which time they were induced with 1 mM isopropylβ-D-thiogalactoside. The bacteria were allowed to express the protein atroom temperature for 6 to 12 hr, then overnight at 4° C., then werepelleted by centrifugation, resuspended in phosphate buffered saline (pH7.4), and lysed in a French press. Bacterial lysates were cleared bycentrifugation at 30,000× g for 30 min. The proteins in the clearedlysates were affinity-purified on Ni-NTA-agarose (Qiagen).

[0137] All GFPs used in these experiments were 238 amino acids inlength. Subcloning the cDNAs encoding the GFPs into PRSET_(B) resultedin the fusion of an additional 33 amino acids to the N-terminus of theGFPs. The sequence of this tag is MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDP (SEQID NO: 21). Thus, the total length of the EYFP-V68L/Q69K (SEQ ID NO: 10)mutants expressed from this cDNA was 271 amino acids. The His₆ tag wasremoved using EKMax (Invitrogen) to determine if the associativeproperties measured for the GFPs were affected by the presence of theN-terminal His₆-tag. A dilution series of the enzyme and His₆-tagged GFPwas made to determine the conditions necessary for complete removal ofthe His₆-tag. The purity of all expressed and purified proteins wasanalyzed by SDS-PAGE. In all cases, the expressed proteins were verypure, with no significant detectable contaminating proteins, and allwere of the proper molecular weight. In addition, removal of the His₆tag was very efficient, as determined by the presence of a single bandmigrating at the lower molecular weight than the His₆-EYFP-V68L/Q69K.

[0138] Spectrophotometric analysis of the purified proteins determinedthat there was no significant change in either the extinctioncoefficient as measured by chromophore denaturation (Ward, supra, 1998)or quantum yield (the standard used for EYFP-V68L/Q69K and the mutantsderived therefrom was fluorescein) of these proteins with respect toEYFP-V68L/Q69K (SEQ ID NO: 10; “wtEYFP”; Table 2). Fluorescence spectrawere taken with a Fluorolog spectrofluorimeter. Absorbance spectra ofproteins were taken with a Cary UV-Vis spectrophotometer. Extinctioncoefficients were determined by the denatured chromophore method (Ward,supra, 1998). TABLE 2 Quantum Extinction Protein Yield CoefficientWtEYFP 0.71*  62,000* His₆ wtEYFP 0.67 67,410 His₆ wtEYFP L221K 0.6764,286 His₆ wtEYFP F223R 0.53 65,393 His₆ wtEYFP A206K 0.62 79,183

[0139] To determine the degree of homoaffinity of the dimers, wtEYFP andthe dimer mutants derived therefrom were subjected to sedimentationequilibrium analytical ultracentrifugation. Purified, recombinantproteins were dialyzed extensively against phosphate buffered saline (pH7.4), and 125 μl samples of protein at concentrations ranging from 50 μMto 700 μM were loaded into 6-channel centrifugation cells with EPONcenterpieces. Samples were blanked against the corresponding dialysisbuffer. Sedimentation equilibrium experiments were performed on aBeckman Optima XL-I analytical ultracentrifuge at 20° C. measuringradial absorbance at 514 nm. Each sample was examined at three or moreof the following speeds: 8,000 rpm, 10,000 rpm, 14,000 rpm, and 20,000rpm. Periodic absorbance measurements at each speed ensured that thesamples had reached equilibrium at each speed.

[0140] The data were analyzed globally at all rotor speeds by nonlinearleast-squares analysis using the software package (Origin) supplied byBeckman. The goodness of fit was evaluated on the basis of the magnitudeand randomness of the residuals, expressed as the difference between theexperimental data and the theoretical curve and also by checking each ofthe tit parameters for physical reasonability. The molecular weight andpartial specific volume of each protein were determined using Sedenterpv 1.01, and the data were factored into the equation for thedetermination of homoaffinity (Table 3). In addition, dissociationconstants (K_(d)) derived from the data generated by analyticalultracentrifugation are shown for some proteins (Table 4). TABLE 3Mutant Molecular Weight Partial Specific Volume wtEYFP 26796.23 0.7332His₆ wtEYFP 30534.26 0.7273 His₆ EYFP A206K 30593.37 0.7277 EYFP L221K30551.29 0.7270 His₆ EYFP L221K 30549.27 0.7271 His₆ EYFP F223R 30543.270.7270 His₆ EYFP L221K/F223R 30560.30 0.7267

[0141] TABLE 4 Protein K_(d) (mM) His₆ wtEYFP 0.11 His₆ wtEYFP L221K 9.7His₆ wtEYFP F223R 4.8 His₆ wtEYFP A206K 74 His₆ wtEYFP L221K/F223R 2.4

[0142] For experiments in living cells, ECFP (SEQ ID NO: 6; “wtECFP”)and EYFP-V68L/Q69K (SEQ ID NO: 10; “wtEYFP”) targeted to the plasmamembrane (PM) were subcloned into the mammalian expression vector,pcDNA3 (Invitrogen Corp.) and mutagenized and sequenced as describedabove. Targeting of the GFP variants to the PM was accomplished bymaking either N-terminal or C-terminal fusions of the GFP variant toshort peptides containing a consensus sequence for acylation and/orprenylation (post-translational lipid modifications). The cDNAs of thePM targeted GFP variants were transfected and expressed in either HeLacells or MDCK cells, and the expression pattern and degree ofassociation were determined using fluorescent microscopy. FRETefficiency was measured to determine the degree of interaction of thePM-ECFP and PM-EYFP-V68L/Q69K. Analysis of the interactions by the FRETdonor-dequench method (Miyawaki and Tsien, supra, 2000) demonstratedthat the wtECFP and wtEYFP interacted in a manner that was dependentupon the association of the wtECFP and wtEYFP, and that this interactionwas effectively eliminated by changing the amino acids in thehydrophobic interface to any one or a combination of the mutationsA206K, L221K and F223R.

[0143] These results demonstrate that the solution oligomeric state ofAequorea GFP and its spectral variants, and dimer mutants derivedtherefrom, were accurately determined by analytical ultracentrifugation.The ECFP (SEQ ID NO: 6) and EYFP-V68L/Q69K (SEQ ID NO: 10) GFP spectralvariants formed homodimers with a fairly high affinity of about 113 μM.By using site directed mutagenesis, the amino acid composition wasaltered so as to effectively eliminate dimerization and the cellbiological problems associated with it. Thus, the modified fluorescentproteins provide a means to use FRET to measure the associativeproperties of host proteins fused to the modified CFP or YFP. Theambiguity and potential for false positive FRET results associated withECFP (SEQ ID NO: 6) and EYFP-V68L/Q69K (SEQ ID NO: 10) dimerization havebeen effectively eliminated, as has the possibility of misidentificationof the subcellular distribution or localization of a host protein due todimerization of GFPs.

[0144] The Renilla GFP and the Discosoma red fluorescent protein (seeExample 2) are obligate oligomers in solution. Because it was generallybelieved that Aequorea GFP could also dimerize in solution, and becauseGFP crystallizes as a dimer, the present investigation was designed tocharacterize the oligomeric state of GFP. The crystallographic interfacebetween the two monomers included many hydrophilic contacts as well asseveral hydrophobic contacts (Yang et al., supra, 1996). It was notimmediately clear, however, to what degree each type of interactioncontributed to the formation of the dimer in solution.

[0145] As disclosed herein, the extent of GFP self-association wasexamined using sedimentation equilibrium, analyticalultracentrifugation, which is very useful for determining the oligomericbehavior of molecules both similar (self associating homomericcomplexes) and dissimilar (heteromeric complexes; see Laue and Stafford,Ann. Rev. Biophys. Biomol. Struct. 28:75-100, 1999). In contrast toX-ray crystallography, the experimental conditions used in theanalytical ultracentrifugation experiments closely approximated cellularphysiological conditions. Monomer contact sites identified by X-raycrystallography within a multimeric complex are not necessarily the sameas those in solution. Also in contrast to analyticalultracentrifugation, X-ray crystallography alone cannot providedefinitive information about the affinity of the complex. The results ofthis investigation demonstrate that replacement of the hydrophobicresidues A206, L221 and F223 with residues containing positively chargedside chains (A206K, L221K and F223R) eliminated dimerization asdetermined by analytical ultracentrifugation in vitro and by analysis ofthe concentration dependence of FRET in intact cells.

EXAMPLE 2 Characterization of the Coral Red Fluorescent Protein, DsRed,and Mutants Thereof

[0146] This example describes the biochemical and biologicalcharacterization of DsRed and DsRed mutants.

[0147] The coding sequence for DsRed was amplified from pDsRed-N1(Clontech Laboratories) with PCR primers that added an N terminal Bam HIrecognition site upstream of the initiator Met codon and a C terminalEco RI site downstream of the STOP codon. After restriction digestion,the PCR product was cloned between the Bam HI and Eco RI sites ofPRSET_(B) (Invitrogen), and the resulting vector was amplified in DH5αbacteria. The resulting plasmid was used as a template for error-pronePCR (Heim and Tsien, Curr. Biol. 6:178-182, 1996, which is incorporatedherein by reference) using primers that were immediately upstream anddownstream of the DsRed coding sequence, theoretically allowing mutationof every coding base, including the initiator Met. The mutagenized PCRfragment was digested with Eco RI and Bam HI and recloned intoPRSET_(B). Alternatively, the Quick-Change mutagenesis kit (Stratagene)was used to make directed mutations on the pRSET_(B)-DsRed plasmid.

[0148] In both random and directed mutagenesis studies, the mutagenizedplasmid library was electroporated into JM109 bacteria, plated on LBplates containing ampicillin, and screened on a digital imaging device(Baird et al., Proc. Natl. Acad. Sci., USA 96:11242-11246, 1999, whichis incorporated herein by reference). This device illuminated plateswith light from a 150 Watt xenon arc lamp, filtered through bandpassexcitation filters and directed onto the plates with two fiber opticbundles. Fluorescence emission from the plates was imaged throughinterference filters with a cooled CCD camera. Images taken at differentwavelengths could be digitally ratioed using MetaMorph software(Universal Imaging) to allow identification of spectrally shiftedmutants. Once selected, the mutant colonies were picked by hand intoLB/Amp medium, after which the culture was used for protein preparationor for plasmid preparations. The DsRed mutant sequences were analyzedwith dye-terminator dideoxy sequencing.

[0149] DsRed and its mutants were purified using the N-terminalpolyhistidine tag (SEQ ID NO: 21; see Example 1) provided by thePRSET_(B) expression vector (see Baird et al., supra, 1999). Theproteins were microconcentrated and buffer exchanged into 10 mM Tris (pH8.5) using a Microcon-30 (Amicon) for spectroscopic characterization.Alternatively, the protein was dialyzed against 10 mM Tris (pH 7.5) foroligomerization studies because microconcentration resulted in theproduction of large protein aggregates. To test for light sensitivity ofprotein maturation, the entire synthesis was repeated in the dark, withculture flasks wrapped in foil, and all purification was performed in aroom that was dimly lit with red lights. There was no difference inprotein yield or color when the protein was prepared in light or dark.

[0150] Numbering of amino acids conforms to the wild type sequence ofdrFP583 (DsRed; Matz et al., supra, 1999), in which residues 66-68,Gln-Tyr-Gly, are homologous to the chromophore-forming residues (65-67,Ser-Tyr-Gly) of GFP. The extra amino acid introduced by Clontech afterthe initiator Met was numbered “1a” and the residues of the N-terminalpolyhistidine tag were numbered ⁻33 to ⁻1.

[0151] Fluorescence spectra were taken with a Fluorologspectrofluorimeter. Absorbance spectra of proteins were taken with aCary UV-Vis spectrophotometer. For quantum yield determination, thefluorescence of a solution of DsRed or DsRed K83M in phosphate bufferedsaline was compared to equally absorbing solutions of Rhodamine B andRhodamine 101 in ethanol. Corrections were included in the quantum yieldcalculation for the refractive index difference between ethanol andwater. For extinction coefficient determination, native proteinabsorbance was measured with the spectrophotometer, and proteinconcentration was measured by the BCA method (Pierce).

[0152] The pH sensitivity of DsRed was determined in a 96 well format byadding 100 μl of dilute DsRed in a weakly buffered solution to 100 μl ofstrongly buffered pH solutions in triplicate (total 200 μl per well) forpH 3 to pH 12. The fluorescence of each well was measured using a525-555 nm bandpass excitation filter and a 575 nm long pass emissionfilter. After the 96 well fluorimeter measurements were taken, 100 μl ofeach pH buffered DsRed solution was analyzed on the spectrofluorimeterto observe pH-dependent spectral shape changes. For time-trials of DsRedmaturation, a dilute solution of freshly synthesized and purified DsRedwas made in 10 mM Tris (pH 8.5), and this solution was stored at roomtemperature in a stoppered cuvette (not airtight) and subjected toperiodic spectral analysis. For mutant maturation data, fluorescenceemission spectra (excitation at 475 nm or 558 nm) were taken directlyafter synthesis and purification, and then after more than 2 monthsstorage at 4° C. or at room temperature.

[0153] Quantum yields for photodestruction were measured separately on amicroscope stage or in a spectrofluorimeter. Microdroplets of aqueousDsRed solution were created under oil on a microscope slide and bleachedwith 1.2 W/cm² of light through a 525-555 nm bandpass filter.Fluorescence over time was monitored using the same filter and a 563-617nm emission filter. For comparison, EGFP (containing mutations F64L,S65T; SEQ ID NO: 6) and EYFP-V68L/Q69K (also containing mutations S65G,S72A, T203Y; SEQ ID NO: 10) microdroplets were similarly bleached with1.9 W/cm² at 460-490 nm while monitoring at 515-555 and 523-548 nm,respectively.

[0154] For the spectrofluorimeter bleaching experiment, a solution ofDsRed was prepared in a rectangular microcuvette and overlaid with oilso that the entire 50 μl of protein solution resided in the 0.25 cm×0.2cm×1 cm illumination volume. The protein solution was illuminated with0.02 W/cm² light from the monochromator centered at 558 nm (5 nmbandwidth). Fluorescence over time was measured at 558 nm excitation(1.25 nm bandwidth) and 583 nm emission. Quantum yields (Φ) forphotobleaching were deduced from the equation Φ=(ε·I·t_(90%))⁻¹, where εis the extinction coefficient in cm²mol⁻¹, I is the intensity ofincident light in einsteins cm⁻²s⁻¹ and t_(90%) is the time in secondsfor the fluorophore to be 90% bleached (Adams et al., J. Am. Chem. Soc.110:3312-3320, 1988, which is incorporated herein by reference).

[0155] Polyhistidine-tagged DsRed, DsRed K83M and wild type Aequorea GFP(SEQ ID NO: 2) were run on a 15% polyacrylamide gel withoutdenaturation. To prevent denaturation, protein solutions (in 10 mM TrisHCl, pH 7.5) were mixed 1:1 with 2× SDS-PAGE sample buffer (containing200 mM dithiothreitol) and loaded directly onto the gel without boiling.A broad range pre-stained molecular weight marker set (BioRad) was usedas a size standard. The gel was then imaged on an Epson 1200 Perfectionflatbed scanner.

[0156] Purified recombinant DsRed was dialyzed extensively againstphosphate buffered saline (pH 7.4) or 10 mM Tris, 1 mM EDTA (pH 7.5).Sedimentation equilibrium experiments were performed on a Beckman OptimaXL-I analytical ultracentrifuge at 20° C. measuring absorbance at 558 nmas a function of radius. 125 μl samples of DsRed at 3.57 μM (0.25absorbance units) were loaded into 6 channel cells. The data wereanalyzed globally at 10,000, 14,000, and 20,000 rpm by nonlinearleast-squares analysis using the Origin software package (Beckman). Thegoodness of fit was evaluated on the basis of the magnitude andrandomness of the residuals, expressed as the difference between theexperimental data and the theoretical curve and also by checking each ofthe fit parameters for physical reasonability.

[0157] FRET between immature green and mature red DsRed was examined inmammalian cells. DsRed in the vector pcDNA3 was transfected into HeLacells using Lipofectin, and 24 hr later the cells were imaged on afluorescence microscope. The fluorescences of the immature green species(excitation 465-495 nm, 505 nm dichroic, emission 523-548 nm) and ofmature red protein (excitation 529-552 nm, 570 nm dichroic, emission563-618 nm) were measured with a cooled CCD camera. These measurementswere repeated after selective photobleaching of the red component byillumination with light from the xenon lamp, filtered only by the 570 nmdichroic, for cumulative durations of 3, 6, 12, 24, and 49 min. By thefinal time, about 95% of the initial red emission had disappeared,whereas the green emission was substantially enhanced.

[0158] Yeast two hybrid assays were also performed. The DsRed codingregion was cloned in-frame downstream of the Gal4 activation domains(the “bait”; amino acid residues 768-881) and DNA binding domains (the“prey”; amino acid residues 1-147) in the pGAD GH and pGBT9 vectors,respectively (Clontech). These DsRed two hybrid plasmids weretransformed into the HF7C strain of S. cerevisiae, which cannotsynthesize histidine in the absence of interaction between the proteinsfused to the Gal4 fragments. Yeast containing both DsRed-bait andDsRed-prey plasmids were streaked on medium lacking histidine andassayed for growth by visually inspecting the plates. Alternatively, theyeast were grown on filters placed on plates lacking tryptophan andleucine to select for the bait and prey plasmids. After overnightgrowth, the filters were removed from the plates, frozen in liquidnitrogen, thawed, and incubated in X-gal overnight at 30° C. and twodays at 4° C. to test for β-galactosidase activity (assayed by bluecolor development). In both the β-galactosidase and histidine growthassays, negative controls consisted of yeast containing bait and preyplasmids, but only the bait or the prey was fused to DsRed.

[0159] Surprisingly, DsRed took days at room temperature to reach fullred fluorescence. At room temperature, a sample of purified proteininitially showed a major component of green fluorescence (excitation andemission maxima at 475 and 499 nm, respectively), which peaked inintensity at about 7 hr and decreased to nearly zero over two days.Meanwhile, the red fluorescence reached half its maximal fluorescenceafter approximately 27 hr and required more than 48 hr to reach greaterthan 90% of maximal fluorescence (see Baird et al., supra, 2000).

[0160] Fully matured DsRed had an extinction coefficient of 75,000M⁻¹cm⁻¹ at its 558 nm absorbance maximum and a fluorescence quantumyield of 0.7, which is much higher than the values of 22,500 M⁻¹cm⁻¹ and0.23 previously reported (Matz et al., supra, 1999). These propertiesmake mature DsRed quite similar to rhodamine dyes in wavelength andbrightness. Unlike most GFP variants, DsRed displayed negligible (<10%)pH-dependence of absorbance or fluorescence from pH 5 to 12. (see Bairdet al., supra, 2000). However, acidification to pH 4-4.5 depressed boththe absorbance and excitation at 558 nm relative to the shorterwavelength shoulder at 526 nm, whereas the emission spectrum wasunchanged in shape. DsRed was also relatively resistant tophotobleaching. When exposed to a beam of 1.2 W/cm² of approximately 540nm light in a microscope stage, microdroplets of DsRed under oil took 1hr to bleach 90%, whereas 20 mW/cm² of 558 nm light in aspectrofluorimeter microcuvette required 83 hr to bleach 90%. Themicroscope and fluorimeter measurements, respectively, gave photobleachquantum efficiencies of 1.06×10⁻⁶ and 4.8×10⁻⁷ (mean of 7.7×10⁻⁷).Analogous microscope measurements of EGFP (S65T; SEQ ID NO: 6) andEYFP-V68L/Q69K (SEQ ID NO: 10; including Q69K) gave 3×10⁻⁶ and 5×10⁻⁵,respectively.

[0161] In an effort to examine the nature of the red chromophore and toidentify DsRed variants useful as biological indicators, DsRed wasmutagenized randomly and at specific sites predicted by sequencealignment with GFP to be near the chromophore. Many mutants that maturedmore slowly or not at all were identified, but none were identified thatmatured faster than DsRed. Screening of random mutants identifiedmutants that appeared green or yellow, which was found to be due tosubstitutions K83E, K83R, S197T, and Y120H. The green fluorescence wasdue to a mutant species with excitation and emission maxima at 475 and500 nm, respectively, whereas the yellow was due to a mixture of thisgreen species with DsRed-like material, rather than to a single speciesat intermediate wavelengths.

[0162] The DsRed K83R mutant had the lowest percentage conversion tored, and proved very useful as a stable version of the immaturegreen-fluorescing form of DsRed (see Baird et al., supra, 2000). Furtherdirected mutagenesis of K83 yielded more green and yellow mutants thatwere impaired in chromophore maturation. In many of the K83 mutants thatmatured slowly and incompletely, the red peak was at longer wavelengthsthan DsRed. K83M was particularly interesting because its finalred-fluorescing species showed a 602 nm emission maximum, withrelatively little residual green fluorescence and a respectable quantumyield, 0.44. However, its maturation was slower than that of the wildtype DsRed. Y120H had a red shift similar to that of K83M and appearedto produce brighter bacterial colonies, but also maintained much moreresidual green fluorescence.

[0163] Spectroscopic data of the DsRed mutants are shown in Table 5.“Maturation” of protein refers to the rate of appearance of the redfluorescence over the two days after protein synthesis. Because somematuration occurs during the synthesis and purification (which take 1-2days), numerical quantification is not accurate. A simple +/− ratingsystem was used, wherein (−−) means very little change, (−) means a 2-5fold increase in red fluorescence, (+) means 5-20 fold increase, and(++) indicates the wild type increase (approximately 40 fold). Thered/green ratio was determined two months after protein synthesis bydividing the peak emission fluorescence obtained at 558 nm excitation bythe 499 nm TABLE 5 Red Species Green Species Maturation Red/GreenMutation Exc (nm) em (nm) exc (nm) Em (nm) Speed Ratio None 558 583 475499 ++ 840 K83R 558 582 480 499 — 0.05 K83E 550 584 474 497 — 0.43 K83N558 592 474 497 − 9.8 K83P 558 594 474 497 − 3.3 K83F 560 594 474 499 —0.29 K83W 562 594 478 501 − 0.44 K83M 564 602 474 499 — 49 Y120H 562 600478 499 − 0.4 S197T 558 584 478 499 + 53 K70R 562 585 480 503 − 13.8K70M N/a n/a 480 499 n/a 0

[0164] fluorescence obtained at 475 nm excitation from the same sample.This does not represent a molar ratio of the two species because theratio does not correct for differences in extinction coefficient orquantum yields between the two species, or the possibility of FRETbetween the two species if they are in a macromolecular complex.

[0165] To determine whether Lys70 or Arg95 can form imines with theterminal carbonyl of a GFP-like chromophore (see Tsien, NatureBiotechnol. 17:956-957, 1999), DsRed mutants K70M, K70R, and R95K wereproduced. K70M remained entirely green with no red component, whereasK70R matured slowly to a slightly red-shifted red species. The spectralsimilarity of K70R to wild type DsRed argues against covalentincorporation of either amino acid into the chromophore. No fluorescenceat any visible wavelength was detected from R95K, which might beexpected because Arg95 is homologous to Arg96 of GFP, which is conservedin all fluorescent proteins characterized to date (Matz et al., supra,1999). The failure of R95K to form a green chromophore prevented testingwhether Arg95 was also required for reddening.

[0166] In view of the propensity of Aequorea GFP to form dimers at highconcentrations in solution and in some crystal forms, and the likelihoodthat Renilla GFP forms an obligate dimer (Ward, supra, 1998), theability of DsRed to oligomerize was examined. Initial examination of theexpressed proteins by SDS-PAGE suggested that aggregates formed, in thatpolyhistidine-tagged proteins DsRed and DsRed K83R migrated as red andyellow-green bands, respectively, at an apparent molecular weight ofgreater than 110 kDa when mixed with 200 mM DTT and not heated beforeloading onto the gel (see Baird et al., supra, 2000). In comparison,Aequorea GFP, when treated similarly, ran as a fluorescent green bandnear its predicted monomer molecular weight of 30 kDa. The highmolecular weight DsRed band was not observed when the sample was brieflyboiled before electrophoresis (see Gross et al., supra, 2000). Underthese conditions, a band near the predicted monomer molecular weight of30 kDa predominated and was colorless without Coomassie staining.

[0167] To determine the oligomerization status more rigorously, theDsRed protein was subjected to analytical equilibrium centrifugation(Laue and Stafford, supra, 1999). Global curve fitting of the absorbancedata determined from the radial scans of equilibrated DsRed indicatedthat DsRed exists as an obligate tetramer in solution (Baird et al.,supra, 2000), in both low salt and physiological salt concentrations.When the data was modeled with a single-species tetramer, the fittedmolecular weight was 119,083 Da, which is in excellent agreement withthe theoretical molecular weight of 119,068 Da for the tetramer ofpolyHis-tagged DsRed. Attempts to fit the curves with alternativestoichiometries from monomer to pentamer failed to converge or gaveunreasonable values for the floating variables and large, non-randomresiduals. The residuals for the tetramer fit were much smaller and morerandomly distributed, but were somewhat further improved by extendingthe model to allow the obligate tetramer to dimerize into an octamer,with a fitted dissociation constant of 39 μM. Thus the 558-nm-absorbingspecies appears to be tetrameric over the range of monomerconcentrations from 14 nM to 11 μM in vitro. The hint of octamerformation at the highest concentrations is only suggestive because thehighest concentrations of tetramer achieved in the ultracentrifugationcell remained more than an order of magnitude below the fitteddissociation constant.

[0168] To confirm whether DsRed also oligomerizes in live cells, FRETanalysis was performed in mammalian cells and in two hybrid assays inyeast cells. HeLa cells were transfected with wild type DsRed and imaged24 hr later, when they contained a mixture of the immature greenintermediate and the final red form. The green fluorescence wasmonitored intermittently before and during selective photobleaching ofthe red species over 49 min of intense orange illumination. If the twoproteins were non-associated, bleaching the red species would beexpected to have no effect on the green fluorescence. In fact, however,the green fluorescence increased by 2.7 to 5.8 fold in different cells,corresponding to FRET efficiencies of 63% to 83%. These values equal orsurpass the highest FRET efficiencies ever observed between GFP mutants,68% for cyan and yellow fluorescent proteins linked by a zincion-saturated zinc finger domain (Miyawaki and Tsien, supra, 2000).

[0169] Additional evidence of in vivo oligomerization was provided bythe directed yeast two hybrid screen. When DsRed fusions to the Gal4 DNAbinding domain and activation domain were expressed in HF7C yeast, theyeast demonstrated a his⁺ phenotype and were able to grow withoutsupplemental histidine, indicating a two hybrid interaction hadoccurred. Neither fusion construct alone (DsRed-DNA binding domain orDsRed-activation domain) produced the his⁺ phenotype, indicating that aDsRed-DsRed interaction, and not a non-specific DsRed-Gal4 interaction,was responsible for the positive result. In addition, the his⁺ yeastturned blue when lysed and incubated with X-gal, suggesting that theDsRed-DsRed interaction also drove transcription of the β-galactosidasegene. Thus, two separate transcriptional measurements of the yeast twohybrid assay confirmed that DsRed associates in vivo.

[0170] This investigation of DsRed revealed a that DsRed as desirableproperties, as well as some nonoptimal properties, with respect to itsbeing useful to complement or as an alternative to GFPs. The mostimportant favorable property identified was that DsRed has a much higherextinction coefficient and fluorescence quantum yield (0.7) than waspreviously reported, such that the fluorescence brightness of the maturewell-folded protein is comparable to rhodamine dyes and to the bestGFPs.

[0171] DsRed also is quite resistant to photobleaching by intensitiestypical of spectrofluorimeters (mW/cm²) or microscopes with arc lampillumination and interference filters (W/cm²), showing a photobleachingquantum yield on the order of 7×10⁻⁷ in both regimes. This value issignificantly better than those for two of the most popular green andyellow GFP mutants, EGFP (3×10⁻⁶) and EYFP-V68L/Q69K (5×10⁻⁵). The meannumber of photons that a single molecule can emit before photobleachingis the ratio of the fluorescence and photobleaching quantum yields, or1×10⁶, 2×10⁵, and 1.5×10⁴ for DsRed, EGFP, and EYFP-V68L/Q69K,respectively. A caveat is that the apparent photobleaching quantum yieldmight well increase at higher light intensities and shorter times if themolecule can be driven into dark states such as triplets or tautomersfrom which it can recover its fluorescence. GFPs usually show a range ofsuch dark states (Dickson et al., Nature 388:355-358, 1997; Schwille etal., Proc. Natl. Acad. Sci., USA 97:151-156, 2000), and there is noreason to expect that DsRed will be any simpler. The photobleachingmeasurements described herein were made over minutes to hours, andinclude ample time for such recovery. In contrast, fluorescencecorrelation spectroscopy and flow cytometry monitor single passages ofmolecules through a focused laser beam within microseconds tomilliseconds, such that temporary dark states that last longer than thetransit time count as photobleaching, raising the apparent quantum yieldfor bleaching. Techniques such as laser scanning confocal microscopy, inwhich identified molecules are repetitively scanned, will showintermediate degrees of photobleaching depending on the time scale ofillumination and recovery.

[0172] Another desirable feature of DsRed is its negligible sensitivityto pH changes over the wide range (pH 4.5 to 12). The currentlyavailable brighter GFP mutants are more readily quenched than DsRed byacidic pH. Such pH sensitivity can be exploited under controlledconditions to sense pH changes, especially inside organelles or otherspecific compartments (see Llopis et al., Proc. Natl. Acad. Sci., USA95:6803-6808, 1998), although this feature can cause artifacts in someapplications.

[0173] DsRed mutants such as K83M demonstrate that DsRed can be pushedto longer wavelengths (564 and 602 nm excitation and emission maxima),while retaining adequate quantum efficiency (0.44). The 6 nm and 19 nmbathochromic shifts correspond to 191 cm⁻¹ and 541 cm⁻¹ in energy, whichare of respectable magnitude for a single amino acid change that doesnot modify the chromophore. A homolog of DsRed recently cloned from asea anemone has an absorbance maximum at 572 nm and extremely weakemission at 595 nm with quantum yield <0.001; one mutant had an emissionpeak at 610 nm but was very dim and slow to mature (Lukyanov et al., J.Biol. Chem. 275:25879-25882, 2000, which is incorporated herein byreference).

[0174] Less desirable features of DsRed include its slow and incompletematuration, and its capacity to oligomerize. A maturation time on theorder of days precludes a use of DsRed as a reporter for short term geneexpression studies and for applications directed to tracking fusionproteins in organisms that have short generation times or fastdevelopment. Since maturation of GFPs was considerably accelerated bymutagenesis (Heim et al., Nature 373:663-664, 1995, which isincorporated herein by reference), DsRed similarly can be mutagenizedand variants having faster maturation times can be isolated.

[0175] Because the Lys83 mutants all permitted at least some maturation,it is unlikely that the primary amine plays a direct catalytic role forthis residue, a suggestion supported by the observation that the mostchemically conservative replacement, Lys to Arg, impeded red developmentto the greatest extent. Ser197 provided a similar result, in that themost conservative possible substitution, Ser to Thr, also significantlyslowed maturation. Mutations at the Lys83 and Ser197 sites appearedseveral times independently in separate random mutagenesis experimentsand, interestingly, Lys83 and Ser197 are replaced by Leu and Thr,respectively, in the highly homologous cyan fluorescent protein dsFP483from the same Discosoma species. Either of the latter two mutationscould explain why dsFP483 never turns red. Residues other than Lys83 andSer197 also affected maturation to the red.

[0176] The multimeric nature of DsRed was demonstrated by four separatelines of evidence, including slow migration on SDS-PAGE unlesspre-boiled, analytical ultracentrifugation, strong FRET from theimmature green to the final red form in mammalian cells, and directedtwo hybrid assays in yeast using HIS3 and LacZ reporter genes.Analytical ultracentrifugation provided the clearest evidence for anobligate stoichiometry of four over the entire range of monomerconcentrations assayed (10⁻⁸ to 10⁻⁵ M), with a hint that octamerformation can occur at yet higher concentrations. In addition, the testsin live cells confirmed that aggregation occurs under typical conditionsof use, including the reducing environment of the cytosol and thepresence of native proteins.

[0177] While oligomerization of DsRed does not preclude its use as areporter of gene expression, it can result in artifactual results inapplications where DsRed is fused to a host protein, for example, toreport on the trafficking or interactions of the host protein in a cell.For a host protein of mass M without its own aggregation tendencies,fusion with DsRed can result in the formation of a complex of at least4(M+26 kDa). Furthermore, since many proteins in signal transduction areactivated by oligomerization, fusion to DsRed and consequent associationcan result in constitutive signaling. For host proteins that areoligomeric, fusion to DsRed can cause clashes of stoichiometry, stericconflicts of quaternary structures, or crosslinking into massiveaggregates. In fact, red cameleons, i.e., fusions of cyan fluorescentprotein, calmodulin, and calmodulin-binding peptide, and DsRed, are farmore prone to form visible punctae in mammalian cells than thecorresponding yellow cameleons with yellow fluorescent protein in placeof DsRed (Miyawaki et al., Proc. Natl. Acad. Sci. USA 96:2135-2140,1999).

[0178] The results disclosed in Example 1, above, indicate that variantsof DsRed, like those of the GFPs, can be produced such that thepropensity of the fluorescent protein to oligomerize is reduced oreliminated. Non-oligomerizing DsRed variants can be constructed andexamined, for example, using a yeast two hybrid or other similar assayto identify and isolate non-aggregating mutants (see Example 1). Inaddition, the X-ray crystallographic structure of DsRed can be examinedto confirm that optimal amino acid residues are modified to produce anon-oligomerizing form of DsRed, and to identify additional residuesthat can be modified so as to reduce or eliminate oligomerization.

EXAMPLE 3 DsRed Variants having Reduced Propensity to Oligomerize

[0179] This example demonstrates that mutations corresponding to thoseintroduced into GFP variants to reduce or eliminate oligomerization alsocan be made in DsRed to reduce the propensity of DsRed to formtetramers.

[0180] In view of the results described in Example 1 and guided by theDsRed crystal structure, amino acid residues were identified aspotentially being involved in DsRed oligomerization. One of these aminoacids, isoleucine-125 (I125), was selected because, in the oligomer, theI125 residues of the subunits were close to each other in a pairwisefashion; i.e., the side chain of I125 of the A subunit was about 4Angstroms from the side chain of I125 of the C subunit, and the I125residues in the B and D subunits were similarly positioned (see FIG. 1).In addition, the area in which the I125 side chains reside exhibitedhydrophobicity, analogous to that identified in Aequorea GFP variants,which was demonstrated to be involved in the inter-subunit interaction(see Example 1). Based on these observations, DsRed mutants containingsubstitutions of positively charged amino acids, Lys (K) and Arg (R),for I125 were generated.

[0181] DsRed I125K and I125R were prepared with the QuickChangeMutagenesis Kit (see Example 1) using the DsRed cDNA (SEQ ID NO: 11;Clontech) subcloned into the expression vector pRSETB (Invitrogen) asthe template for mutagenesis. The primers for mutagenesis, with themutated codons underlined, were as follows: I125K (forward) 5′-TAC AAGGTG AAG TTC AAG GGC GTG (SEQ ID NO:22); AAC TTC CCC-3′; I125K (reverse)5′-GGG GAA GTT CAC GCC CTT GAA CTT (SEQ ID NO:23) CAC CTT GTA-3′; I125R(forward) 5′-TAC AAG GTG AAG TTC CGC GGC GTG (SEQ ID NO:24) AAC TTCCCC-3′; and I125R (reverse) 5′-GGG GAA GTT CAC GCC GCG GAA CTT (SEQ IDNO:25) CAC CTT GTA-3′.

[0182] The mutant proteins were prepared following standard methodologyand analyzed with polyacrylamide gel electrophoresis as described (Bairdet al., supra, 2000). For further analysis, DsRed I125R was dialyzedextensively in PBS, then diluted in PBS until the absorbance of thesolution at 558 nm was 0.1. This solution was centrifuged in a BeckmanXL-1 analytical ultracentrifuge in PBS at 10,000 rpm, 12,000 rpm, 14,000rpm, and 20,000 rpm. Absorbance at 558 nm versus radius was determinedand compared to a wild type tetrameric DsRed control (Baird et al.,supra, 2000).

[0183] The DsRed I125K yielded a protein that became red fluorescent andwas a mixture of dimer and tetramer as analyzed by non-denaturingpolyacrylamide gel electrophoresis of the native protein. The sameanalysis of Ds Red I125R revealed that the protein was entirely dimeric.The dimeric status of DsRed I125R was confirmed by analyticalultracentrifugation; no residual tetramer was detected. These resultsdemonstrate that the interaction between the A:C subunits and the B:Dsubunits can be disrupted, thereby reducing the propensity of the DsRedvariant to oligomerize (see FIG. 1). No attempt was made to disrupt theA:B and C:D interfaces. These results demonstrate that the method ofreducing or eliminating oligomerization of the GFP variants as describedin Example 1 is generally applicable to other fluorescent proteins thathave a propensity to oligomerize.

EXAMPLE 4 Preparation and Characterization of Non-Oligomerizing TandemDsRed

[0184] This example demonstrates that a tandem DsRed protein can beformed by linking two DsRed monomers, and that such tandem DsRedproteins maintain emission and excitation spectra characteristic ofDsRed, but do not oligomerize.

[0185] To construct tDsRed, a 3′ primer,5′-CCGGATCCCCTTTGGTGCTGCCCTCTCCGCTGCCAGGCTTGCCGCTGCCGCTGGTGCTGCCAAGGAACAGATGGTGGCGTCCCTCG-3′ (SEQ ID NO:27), was designedthat overlapped the last 25 bp of DsRed (derived from the Clontechvector pDsRed-N1) and encoded for the linker sequence GSTSGSGKPGSGEGSTKG(SEQ ID NO:26), followed by a Bam HI restriction site in frame with theBam HI site of PRSET_(B) (Invitrogen). It was later determined that theabove primer sequence contains three mismatches in the overlap regionand contained several codons that were not optimal for mammalianexpression. Accordingly, a new 3′ primer,5′-CCGGATCCCCCTTGGTGCTGCCCTCCCCGCTGCCGGGCTTCCCGCTCCCGCTGGTGCTGCCCAGGAACAGGTGGTGGCGGCCCTCG-3′ (SEQ ID NO:28), also wasused. The 5′ primer, 5′-GTACGA CGATGACGATAAGGATCC-3′ (SEQ ID NO:29) alsocontained a Bam HI restriction site in frame with the Bam HI site ofPRSET_(B).

[0186] PCR amplification of DsRed and of DsRed-I125R with the new linkerwas accomplished with Taq DNA polymerase (Roche) and an annealingprotocol that included 2 cycles at 40° C., 5 cycles at 43° C., 5 cyclesat 45° C., and 15 cycles at 52° C. The resulting PCR product waspurified by agarose gel-electrophoresis and digested with Bam HI (NewEngland Biolabs). Bam HI and calf intestinal phosphatase (New EnglandBiolabs) treated vector was prepared from PRSET_(B) with DsRed orDsRed-I125R inserted in frame with the His-6 tag and between the 5′ BamHI and 3′ Eco RI restriction sites.

[0187] Following ligation of the digested PCR products and vector withT4 DNA ligase (NEB), the mixture was used to transform competent E. coliDH5α by heat shock. Transformed colonies were grown on LB agar platessupplemented with the antibiotic ampicillin. Colonies were picked atrandom, and plasmid DNA was isolated through standard miniprepprocedures (Qiagen). DNA sequencing was used to confirm the correctorientation of the inserted sequence.

[0188] In order to express protein, the isolated and sequenced vectorswere used to transform competent E. coli JM109(DE3). Single coloniesgrown on LB agar/ampicillin were used to inoculate 1 liter cultures ofLB/ampicillin, then were grown with shaking at 225 rpm and 37° C. untilthe broth reached an OD₆₀₀ of 0.5-1.0. IPTG was added to a finalconcentration of 100 mg/l and the culture was grown for either 5 hr at37° C. (tDsRed) or 24 hr at room temperature (RT; tDsRed-I125R). Cellswere harvested by centrifugation (10 min, 5000 rpm), the pellet wasresuspended in 50 mM Tris pH 7.5, and the cells were lysed by a singlepass through a French press. Protein was purified by Ni-NTA (Qiagen)chromatography as described by the manufacturer and was stored in theelution buffer or was dialyzed into 50 mM Tris, pH 7.5.

[0189] With respect to the excitation and emission spectra as well asthe maturation time of tDsRed and tDsRed-I125R, the proteins behavedidentically to their untethered counterparts. As expected, tDsReddeveloped visible fluorescence within approximately 12 hr at RT, whiletDsRed-I125R required several days before significant red colordeveloped. The maturation of tDsRed-I125R continued for up toapproximately 10 days. The excitation and emission maxima were unchangedat 558 nm and 583 nm, respectively.

[0190] The differences in the tandem dimer became apparent when theproteins were analyzed by SDS-polyacrylamide electrophoresis. Due to thehigh stability of the tetramer, DsRed that was not subjected to boilingmigrated with an apparent molecular mass of about 110 kDa. In addition,the band on the gel, which corresponded to a DsRed tetramer, retainedits red fluorescence, indicating that the rigid barrel structure of eachmonomer was intact. When the sample was boiled before loading, DsRed wasnon-fluorescent and presumably denatured, and ran as a monomer ofapproximately 32 kDa.

[0191] SDS-PAGE analysis confirmed the tandem structure of the expressedred fluorescent proteins, tDsRed and tDsRed-I125R. The unboiled tDsRedmigrated at the same apparent molecular mass (about 110 kDa) as unboilednormal DsRed. The difference in their molecular structures only wasapparent when the samples were boiled (denatured) before they wereloaded onto the gel. Boiled tDsRed migrated with an apparent molecularmass of about 65 kDa, which is approximately the mass of two DsRedmonomers, whereas boiled DsRed migrated at the monomer molecular mass of32 kDa.

[0192] A similar comparison was made for DsRed-1125R and tDsRed-I125R.When they were not boiled prior to SDS-PAGE, tDsRed-I125R andDsRed-I125R both migrated as dimers with an apparent molecular mass ofabout 50 kDa. DsRed-I125R that was not boiled also had a large componentthat appeared to be denatured, though the fluorescent band for the dimer(50 kDa) was clearly visible. tDsRed-I125R also had a denaturedcomponent that migrated slower (65 kDa vs. 50 kDa) than the intactfluorescent species. However, when boiled, tDsRed-I125R migrated atapproximately the same mass as two monomers (65 kDa), while DsRed-1125Rmigrated at the monomer molecular mass of 32 kDa.

[0193] These results demonstrate that linking two DsRed monomers to forman intramolecularly bound tandem dimer prevented formation ofintermolecular oligomers, without affecting the emission or excitationspectra of the red fluorescent proteins.

[0194] Although the invention has been described with reference to theabove examples, it will be understood that modifications and variationsare encompassed within the spirit and scope of the invention.Accordingly, the invention is limited only by the following claims.

1 29 1 716 DNA Aequorea victoria CDS (1)..(714) 1 atg agt aaa gga gaagaa ctt ttc act gga gtt gtc cca att ctt gtt 48 Met Ser Lys Gly Glu GluLeu Phe Thr Gly Val Val Pro Ile Leu Val 1 5 10 15 gaa tta gat ggt gatgtt aat ggg cac aaa ttt tct gtc agt gga gag 96 Glu Leu Asp Gly Asp ValAsn Gly His Lys Phe Ser Val Ser Gly Glu 20 25 30 ggt gaa ggt gat gca acatac gga aaa ctt acc ctt aaa ttt att tgc 144 Gly Glu Gly Asp Ala Thr TyrGly Lys Leu Thr Leu Lys Phe Ile Cys 35 40 45 act act gga aaa cta cct gttcca tgg cca aca ctt gtc act act ttc 192 Thr Thr Gly Lys Leu Pro Val ProTrp Pro Thr Leu Val Thr Thr Phe 50 55 60 tct tat ggt gtt caa tgc ttt tcaaga tac cca gat cat atg aaa cag 240 Ser Tyr Gly Val Gln Cys Phe Ser ArgTyr Pro Asp His Met Lys Gln 65 70 75 80 cat gac ttt ttc aag agt gcc atgccc gaa ggt tat gta cag gaa aga 288 His Asp Phe Phe Lys Ser Ala Met ProGlu Gly Tyr Val Gln Glu Arg 85 90 95 act ata ttt ttc aaa gat gac ggg aactac aag aca cgt gct gaa gtc 336 Thr Ile Phe Phe Lys Asp Asp Gly Asn TyrLys Thr Arg Ala Glu Val 100 105 110 aag ttt gaa ggt gat acc ctt gtt aataga atc gag tta aaa ggt att 384 Lys Phe Glu Gly Asp Thr Leu Val Asn ArgIle Glu Leu Lys Gly Ile 115 120 125 gat ttt aaa gaa gat gga aac att cttgga cac aaa ttg gaa tac aac 432 Asp Phe Lys Glu Asp Gly Asn Ile Leu GlyHis Lys Leu Glu Tyr Asn 130 135 140 tat aac tca cac aat gta tac atc atggca gac aaa caa aag aat gga 480 Tyr Asn Ser His Asn Val Tyr Ile Met AlaAsp Lys Gln Lys Asn Gly 145 150 155 160 atc aaa gtt aac ttc aaa att agacac aac att gaa gat gga agc gtt 528 Ile Lys Val Asn Phe Lys Ile Arg HisAsn Ile Glu Asp Gly Ser Val 165 170 175 caa cta gca gac cat tat caa caaaat act cca att ggc gat ggc cct 576 Gln Leu Ala Asp His Tyr Gln Gln AsnThr Pro Ile Gly Asp Gly Pro 180 185 190 gtc ctt tta cca gac aac cat tacctg tcc aca caa tct gcc ctt tcg 624 Val Leu Leu Pro Asp Asn His Tyr LeuSer Thr Gln Ser Ala Leu Ser 195 200 205 aaa gat ccc aac gaa aag aga gaccac atg gtc ctt ctt gag ttt gta 672 Lys Asp Pro Asn Glu Lys Arg Asp HisMet Val Leu Leu Glu Phe Val 210 215 220 aca gct gct ggg att aca cat ggcatg gat gaa cta tac aaa ta 716 Thr Ala Ala Gly Ile Thr His Gly Met AspGlu Leu Tyr Lys 225 230 235 2 238 PRT Aequorea victoria 2 Met Ser LysGly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val 1 5 10 15 Glu LeuAsp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu 20 25 30 Gly GluGly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys 35 40 45 Thr ThrGly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe 50 55 60 Ser TyrGly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln 65 70 75 80 HisAsp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 85 90 95 ThrIle Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val 100 105 110Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile 115 120125 Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn 130135 140 Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly145 150 155 160 Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp GlySer Val 165 170 175 Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile GlyAsp Gly Pro 180 185 190 Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr GlnSer Ala Leu Ser 195 200 205 Lys Asp Pro Asn Glu Lys Arg Asp His Met ValLeu Leu Glu Phe Val 210 215 220 Thr Ala Ala Gly Ile Thr His Gly Met AspGlu Leu Tyr Lys 225 230 235 3 720 DNA Aequorea victoria CDS (1)..(720) 3atg gtg agc aag ggc gag gag ctg ttc acc ggg gtg gtg ccc atc ctg 48 MetVal Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15gtc gag ctg gac ggc gac gta aac ggc cac aag ttc agc gtg tcc ggc 96 ValGlu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30 gagggc gag ggc gat gcc acc tac ggc aag ctg acc ctg aag ttc atc 144 Glu GlyGlu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 tgc accacc ggc aag ctg ccc gtg ccc tgg ccc acc ctc gtg acc acc 192 Cys Thr ThrGly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 ctg acc tacggc gtg cag tgc ttc agc cgc tac ccc gac cac atg aag 240 Leu Thr Tyr GlyVal Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys 65 70 75 80 cag cac gacttc ttc aag tcc gcc atg ccc gaa ggc tac gtc cag gag 288 Gln His Asp PhePhe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 cgc acc atc ttcttc aag gac gac ggc aac tac aag acc cgc gcc gag 336 Arg Thr Ile Phe PheLys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 gtg aag ttc gagggc gac acc ctg gtg aac cgc atc gag ctg aag ggc 384 Val Lys Phe Glu GlyAsp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 atc gac ttc aaggag gac ggc aac atc ctg ggg cac aag ctg gag tac 432 Ile Asp Phe Lys GluAsp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 aac tac aac agccac aac gtc tat atc atg gcc gac aag cag aag aac 480 Asn Tyr Asn Ser HisAsn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 145 150 155 160 ggc atc aaggtg aac ttc aag atc cgc cac aac atc gag gac ggc agc 528 Gly Ile Lys ValAsn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 165 170 175 gtg cag ctcgcc gac cac tac cag cag aac acc ccc atc ggc gac ggc 576 Val Gln Leu AlaAsp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 180 185 190 ccc gtg ctgctg ccc gac aac cac tac ctg agc acc cag tcc gcc ctg 624 Pro Val Leu LeuPro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu 195 200 205 agc aaa gacccc aac gag aag cgc gat cac atg gtc ctg ctg gag ttc 672 Ser Lys Asp ProAsn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 210 215 220 gtg acc gccgcc ggg atc act ctc ggc atg gac gag ctg tac aag taa 720 Val Thr Ala AlaGly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys 225 230 235 4 239 PRTAequorea victoria 4 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val ValPro Ile Leu 1 5 10 15 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys PheSer Val Ser Gly 20 25 30 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu ThrLeu Lys Phe Ile 35 40 45 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro ThrLeu Val Thr Thr 50 55 60 Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr ProAsp His Met Lys 65 70 75 80 Gln His Asp Phe Phe Lys Ser Ala Met Pro GluGly Tyr Val Gln Glu 85 90 95 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn TyrLys Thr Arg Ala Glu 100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val AsnArg Ile Glu Leu Lys Gly 115 120 125 Ile Asp Phe Lys Glu Asp Gly Asn IleLeu Gly His Lys Leu Glu Tyr 130 135 140 Asn Tyr Asn Ser His Asn Val TyrIle Met Ala Asp Lys Gln Lys Asn 145 150 155 160 Gly Ile Lys Val Asn PheLys Ile Arg His Asn Ile Glu Asp Gly Ser 165 170 175 Val Gln Leu Ala AspHis Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 180 185 190 Pro Val Leu LeuPro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu 195 200 205 Ser Lys AspPro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 210 215 220 Val ThrAla Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys 225 230 235 5 720DNA Aequorea victoria CDS (1)..(720) 5 atg gtg agc aag ggc gag gag ctgttc acc ggg gtg gtg ccc atc ctg 48 Met Val Ser Lys Gly Glu Glu Leu PheThr Gly Val Val Pro Ile Leu 1 5 10 15 gtc gag ctg gac ggc gac gta aacggc cac agg ttc agc gtg tcc ggc 96 Val Glu Leu Asp Gly Asp Val Asn GlyHis Arg Phe Ser Val Ser Gly 20 25 30 gag ggc gag ggc gat gcc acc tac ggcaag ctg acc ctg aag ttc atc 144 Glu Gly Glu Gly Asp Ala Thr Tyr Gly LysLeu Thr Leu Lys Phe Ile 35 40 45 tgc acc acc ggc aag ctg ccc gtg ccc tggccc acc ctc gtg acc acc 192 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp ProThr Leu Val Thr Thr 50 55 60 ctg acc tgg ggc gtg cag tgc ttc agc cgc tacccc gac cac atg aag 240 Leu Thr Trp Gly Val Gln Cys Phe Ser Arg Tyr ProAsp His Met Lys 65 70 75 80 cag cac gac ttc ttc aag tcc gcc atg ccc gaaggc tac gtc cag gag 288 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu GlyTyr Val Gln Glu 85 90 95 cgt acc atc ttc ttc aag gac gac ggc aac tac aagacc cgc gcc gag 336 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys ThrArg Ala Glu 100 105 110 gtg aag ttc gag ggc gac acc ctg gtg aac cgc atcgag ctg aag ggc 384 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile GluLeu Lys Gly 115 120 125 atc gac ttc aag gag gac ggc aac atc ctg ggg cacaag ctg gag tac 432 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His LysLeu Glu Tyr 130 135 140 aac tac atc agc cac aac gtc tat atc acc gcc gacaag cag aag aac 480 Asn Tyr Ile Ser His Asn Val Tyr Ile Thr Ala Asp LysGln Lys Asn 145 150 155 160 ggc atc aag gcc cac ttc aag atc cgc cac aacatc gag gac ggc agc 528 Gly Ile Lys Ala His Phe Lys Ile Arg His Asn IleGlu Asp Gly Ser 165 170 175 gtg cag ctc gcc gac cac tac cag cag aac accccc atc ggc gac ggc 576 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr ProIle Gly Asp Gly 180 185 190 ccc gtg ctg ctg ccc gac aac cac tac ctg agcacc cag tcc gcc ctg 624 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser ThrGln Ser Ala Leu 195 200 205 agc aaa gac ccc aac gag aag cgc gat cac atggtc ctg ctg gag ttc 672 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met ValLeu Leu Glu Phe 210 215 220 gtg acc gcc gcc ggg atc act ctc ggc atg gacgag ctg tac aag taa 720 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp GluLeu Tyr Lys 225 230 235 6 239 PRT Aequorea victoria 6 Met Val Ser LysGly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 Val Glu LeuAsp Gly Asp Val Asn Gly His Arg Phe Ser Val Ser Gly 20 25 30 Glu Gly GluGly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 Cys Thr ThrGly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 Leu Thr TrpGly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys 65 70 75 80 Gln HisAsp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 Arg ThrIle Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 ValLys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135140 Asn Tyr Ile Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn 145150 155 160 Gly Ile Lys Ala His Phe Lys Ile Arg His Asn Ile Glu Asp GlySer 165 170 175 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile GlyAsp Gly 180 185 190 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr GlnSer Ala Leu 195 200 205 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met ValLeu Leu Glu Phe 210 215 220 Val Thr Ala Ala Gly Ile Thr Leu Gly Met AspGlu Leu Tyr Lys 225 230 235 7 720 DNA Aequorea victoria CDS (1)..(720) 7atg gtg agc aag ggc gag gag ctg ttc acc ggg gtg gtg ccc atc ctg 48 MetVal Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15gtc gag ctg gac ggc gac gta aac ggc cac aag ttc agc gtg tcc ggc 96 ValGlu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30 gagggc gag ggc gat gcc acc tac ggc aag ctg acc ctg aag ttc atc 144 Glu GlyGlu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 tgc accacc ggc aag ctg ccc gtg ccc tgg ccc acc ctc gtg acc acc 192 Cys Thr ThrGly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 ttc ggc tacggc gtg cag tgc ttc gcc cgc tac ccc gac cac atg aag 240 Phe Gly Tyr GlyVal Gln Cys Phe Ala Arg Tyr Pro Asp His Met Lys 65 70 75 80 cag cac gacttc ttc aag tcc gcc atg ccc gaa ggc tac gtc cag gag 288 Gln His Asp PhePhe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 cgc acc atc ttcttc aag gac gac ggc aac tac aag acc cgc gcc gag 336 Arg Thr Ile Phe PheLys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 gtg aag ttc gagggc gac acc ctg gtg aac cgc atc gag ctg aag ggc 384 Val Lys Phe Glu GlyAsp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 atc gac ttc aaggag gac ggc aac atc ctg ggg cac aag ctg gag tac 432 Ile Asp Phe Lys GluAsp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 aac tac aac agccac aac gtc tat atc atg gcc gac aag cag aag aac 480 Asn Tyr Asn Ser HisAsn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 145 150 155 160 ggc atc aaggtg aac ttc aag atc cgc cac aac atc gag gac ggc agc 528 Gly Ile Lys ValAsn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 165 170 175 gtg cag ctcgcc gac cac tac cag cag aac acc ccc atc ggc gac ggc 576 Val Gln Leu AlaAsp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 180 185 190 ccc gtg ctgctg ccc gac aac cac tac ctg agc tac cag tcc gcc ctg 624 Pro Val Leu LeuPro Asp Asn His Tyr Leu Ser Tyr Gln Ser Ala Leu 195 200 205 agc aaa gacccc aac gag aag cgc gat cac atg gtc ctg ctg gag ttc 672 Ser Lys Asp ProAsn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 210 215 220 gtg acc gccgcc ggg atc act ctc ggc atg gac gag ctg tac aag taa 720 Val Thr Ala AlaGly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys 225 230 235 8 239 PRTAequorea victoria 8 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val ValPro Ile Leu 1 5 10 15 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys PheSer Val Ser Gly 20 25 30 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu ThrLeu Lys Phe Ile 35 40 45 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro ThrLeu Val Thr Thr 50 55 60 Phe Gly Tyr Gly Val Gln Cys Phe Ala Arg Tyr ProAsp His Met Lys 65 70 75 80 Gln His Asp Phe Phe Lys Ser Ala Met Pro GluGly Tyr Val Gln Glu 85 90 95 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn TyrLys Thr Arg Ala Glu 100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val AsnArg Ile Glu Leu Lys Gly 115 120 125 Ile Asp Phe Lys Glu Asp Gly Asn IleLeu Gly His Lys Leu Glu Tyr 130 135 140 Asn Tyr Asn Ser His Asn Val TyrIle Met Ala Asp Lys Gln Lys Asn 145 150 155 160 Gly Ile Lys Val Asn PheLys Ile Arg His Asn Ile Glu Asp Gly Ser 165 170 175 Val Gln Leu Ala AspHis Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 180 185 190 Pro Val Leu LeuPro Asp Asn His Tyr Leu Ser Tyr Gln Ser Ala Leu 195 200 205 Ser Lys AspPro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 210 215 220 Val ThrAla Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys 225 230 235 9 720DNA Aequorea victoria CDS (1)..(720) 9 atg gtg agc aag ggc gag gag ctgttc acc ggg gtg gtg ccc atc ctg 48 Met Val Ser Lys Gly Glu Glu Leu PheThr Gly Val Val Pro Ile Leu 1 5 10 15 gtc gag ctg gac ggc gac gta aacggc cac aag ttc agc gtg tcc ggc 96 Val Glu Leu Asp Gly Asp Val Asn GlyHis Lys Phe Ser Val Ser Gly 20 25 30 gag ggc gag ggc gat gcc acc tac ggcaag ctg acc ctg aag ttc atc 144 Glu Gly Glu Gly Asp Ala Thr Tyr Gly LysLeu Thr Leu Lys Phe Ile 35 40 45 tgc acc acc ggc aag ctg ccc gtg ccc tggccc acc ctc gtg acc acc 192 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp ProThr Leu Val Thr Thr 50 55 60 ttc ggc tac ggc ctg aag tgc ttc gcc cgc tacccc gac cac atg aag 240 Phe Gly Tyr Gly Leu Lys Cys Phe Ala Arg Tyr ProAsp His Met Lys 65 70 75 80 cag cac gac ttc ttc aag tcc gcc atg ccc gaaggc tac gtc cag gag 288 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu GlyTyr Val Gln Glu 85 90 95 cgc acc atc ttc ttc aag gac gac ggc aac tac aagacc cgc gcc gag 336 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys ThrArg Ala Glu 100 105 110 gtg aag ttc gag ggc gac acc ctg gtg aac cgc atcgag ctg aag ggc 384 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile GluLeu Lys Gly 115 120 125 atc gac ttc aag gag gac ggc aac atc ctg ggg cacaag ctg gag tac 432 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His LysLeu Glu Tyr 130 135 140 aac tac aac agc cac aac gtc tat atc atg gcc gacaag cag aag aac 480 Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp LysGln Lys Asn 145 150 155 160 ggc atc aag gtg aac ttc aag atc cgc cac aacatc gag gac ggc agc 528 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn IleGlu Asp Gly Ser 165 170 175 gtg cag ctc gcc gac cac tac cag cag aac accccc atc ggc gac ggc 576 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr ProIle Gly Asp Gly 180 185 190 ccc gtg ctg ctg ccc gac aac cac tac ctg agctac cag tcc gcc ctg 624 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser TyrGln Ser Ala Leu 195 200 205 agc aaa gac ccc aac gag aag cgc gat cac atggtc ctg ctg gag ttc 672 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met ValLeu Leu Glu Phe 210 215 220 gtg acc gcc gcc ggg atc act ctc ggc atg gacgag ctg tac aag taa 720 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp GluLeu Tyr Lys 225 230 235 10 239 PRT Aequorea victoria 10 Met Val Ser LysGly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 Val Glu LeuAsp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30 Glu Gly GluGly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 Cys Thr ThrGly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 Phe Gly TyrGly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys 65 70 75 80 Gln HisAsp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 Arg ThrIle Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 ValLys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135140 Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 145150 155 160 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp GlySer 165 170 175 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile GlyAsp Gly 180 185 190 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Tyr GlnSer Ala Leu 195 200 205 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met ValLeu Leu Glu Phe 210 215 220 Val Thr Ala Ala Gly Ile Thr Leu Gly Met AspGlu Leu Tyr Lys 225 230 235 11 859 DNA Discosoma sp. CDS (54)..(731) 11gtttcagcca gtgacggtca gtgacagggt gagccacttg gtataccaac aaa atg 56 Met 1agg tct tcc aag aat gtt atc aag gag ttc atg agg ttt aag gtt cgc 104 ArgSer Ser Lys Asn Val Ile Lys Glu Phe Met Arg Phe Lys Val Arg 5 10 15 atggaa gga acg gtc aat ggg cac gag ttt gaa ata gaa ggc gaa gga 152 Met GluGly Thr Val Asn Gly His Glu Phe Glu Ile Glu Gly Glu Gly 20 25 30 gag gggagg cca tac gaa ggc cac aat acc gta aag ctt aag gta acc 200 Glu Gly ArgPro Tyr Glu Gly His Asn Thr Val Lys Leu Lys Val Thr 35 40 45 aag ggg ggacct ttg cca ttt gct tgg gat att ttg tca cca caa ttt 248 Lys Gly Gly ProLeu Pro Phe Ala Trp Asp Ile Leu Ser Pro Gln Phe 50 55 60 65 cag tat ggaagc aag gta tat gtc aag cac cct gcc gac ata cca gac 296 Gln Tyr Gly SerLys Val Tyr Val Lys His Pro Ala Asp Ile Pro Asp 70 75 80 tat aaa aag ctgtca ttt cct gaa gga ttt aaa tgg gaa agg gtc atg 344 Tyr Lys Lys Leu SerPhe Pro Glu Gly Phe Lys Trp Glu Arg Val Met 85 90 95 aac ttt gaa gac ggtggc gtc gtt act gta acc cag gat tcc agt ttg 392 Asn Phe Glu Asp Gly GlyVal Val Thr Val Thr Gln Asp Ser Ser Leu 100 105 110 cag gat ggc tgt ttcatc tac aag gtc aag ttc att ggc gtg aac ttt 440 Gln Asp Gly Cys Phe IleTyr Lys Val Lys Phe Ile Gly Val Asn Phe 115 120 125 cct tcc gat gga cctgtt atg caa aag aag aca atg ggc tgg gaa gcc 488 Pro Ser Asp Gly Pro ValMet Gln Lys Lys Thr Met Gly Trp Glu Ala 130 135 140 145 agc act gag cgtttg tat cct cgt gat ggc gtg ttg aaa gga gag att 536 Ser Thr Glu Arg LeuTyr Pro Arg Asp Gly Val Leu Lys Gly Glu Ile 150 155 160 cat aag gct ctgaag ctg aaa gac ggt ggt cat tac cta gtt gaa ttc 584 His Lys Ala Leu LysLeu Lys Asp Gly Gly His Tyr Leu Val Glu Phe 165 170 175 aaa agt att tacatg gca aag aag cct gtg cag cta cca ggg tac tac 632 Lys Ser Ile Tyr MetAla Lys Lys Pro Val Gln Leu Pro Gly Tyr Tyr 180 185 190 tat gtt gac tccaaa ctg gat ata aca agc cac aac gaa gac tat aca 680 Tyr Val Asp Ser LysLeu Asp Ile Thr Ser His Asn Glu Asp Tyr Thr 195 200 205 atc gtt gag cagtat gaa aga acc gag gga cgc cac cat ctg ttc ctt 728 Ile Val Glu Gln TyrGlu Arg Thr Glu Gly Arg His His Leu Phe Leu 210 215 220 225 taaggctgaactt ggctcagacg tgggtgagcg gtaatgacca caaaaggcag 781 cgaagaaaaaccatgatcgt tttttttagg ttggcagcct gaaatcgtag gaaatacatc 841 agaaatgttacaaacagg 859 12 225 PRT Discosoma sp. 12 Met Arg Ser Ser Lys Asn Val IleLys Glu Phe Met Arg Phe Lys Val 1 5 10 15 Arg Met Glu Gly Thr Val AsnGly His Glu Phe Glu Ile Glu Gly Glu 20 25 30 Gly Glu Gly Arg Pro Tyr GluGly His Asn Thr Val Lys Leu Lys Val 35 40 45 Thr Lys Gly Gly Pro Leu ProPhe Ala Trp Asp Ile Leu Ser Pro Gln 50 55 60 Phe Gln Tyr Gly Ser Lys ValTyr Val Lys His Pro Ala Asp Ile Pro 65 70 75 80 Asp Tyr Lys Lys Leu SerPhe Pro Glu Gly Phe Lys Trp Glu Arg Val 85 90 95 Met Asn Phe Glu Asp GlyGly Val Val Thr Val Thr Gln Asp Ser Ser 100 105 110 Leu Gln Asp Gly CysPhe Ile Tyr Lys Val Lys Phe Ile Gly Val Asn 115 120 125 Phe Pro Ser AspGly Pro Val Met Gln Lys Lys Thr Met Gly Trp Glu 130 135 140 Ala Ser ThrGlu Arg Leu Tyr Pro Arg Asp Gly Val Leu Lys Gly Glu 145 150 155 160 IleHis Lys Ala Leu Lys Leu Lys Asp Gly Gly His Tyr Leu Val Glu 165 170 175Phe Lys Ser Ile Tyr Met Ala Lys Lys Pro Val Gln Leu Pro Gly Tyr 180 185190 Tyr Tyr Val Asp Ser Lys Leu Asp Ile Thr Ser His Asn Glu Asp Tyr 195200 205 Thr Ile Val Glu Gln Tyr Glu Arg Thr Glu Gly Arg His His Leu Phe210 215 220 Leu 225 13 42 DNA Artificial Sequence PRIMER A206K top 13cagtccaagc tgagcaaaga ccccaacgag aagcgcgatc ac 42 14 42 DNA ArtificialSequence PRIMER A206K bottom 14 gtgatcgcgc ttctcgttgg ggtctttgctcagcttggac tg 42 15 36 DNA Artificial Sequence PRIMER L221K top 15cacatggtcc tgaaggagtt cgtgaccgcc gccggg 36 16 36 DNA Artificial SequencePRIMER L221K bottom 16 cccggcggcg gtcacgaact ccttcaggac catgtg 36 17 36DNA Artificial Sequence PRIMER F223R top 17 cacatggtcc tgctggagcgcgtgaccgcc gccggg 36 18 36 DNA Artificial Sequence PRIMER F223R bottom18 cccggcggcg gtcacgcgct ccagcaggac catgtg 36 19 36 DNA ArtificialSequence PRIMER L221K/F223R top 19 cacatcgtcc tgaaggagcg cgtgaccgccgccggg 36 20 36 DNA Artificial Sequence PRIMER L221K/F223R bottom 20cccggcggcg gtcacgcgct ccttcaggac catgtg 36 21 33 PRT Artificial SequenceADDITIONAL 33 AMINO ACIDS TAG TO THE N-TERMINUS OF THE GFPs 21 Met ArgGly Ser His His His His His His Gly Met Ala Ser Met Thr 1 5 10 15 GlyGly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp 20 25 30 Pro22 33 DNA Artificial Sequence PRIMER I125K 22 tacaaggtga agttcaagggcgtgaacttc ccc 33 23 33 DNA Artificial Sequence PRIMER L125K reverse 23ggggaagttc acgcccttga acttcacctt gta 33 24 33 DNA Artificial SequencePRIMER I125R forward 24 tacaaggtga agttccgcgg cgtgaacttc ccc 33 25 33DNA Artificial Sequence PRIMER I125R reverse 25 ggggaagttc acgccgcggaacttcacctt gta 33 26 18 PRT Artificial Sequence Synthetic peptide 26 GlySer Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr 1 5 10 15Lys Gly 27 86 DNA Artificial Sequence Primer for PCR 27 ccggatcccctttggtgctg ccctctccgc tgccaggctt gccgctgccg ctggtgctgc 60 caaggaacagatggtggcgt ccctcg 86 28 86 DNA Artificial Sequence Primer for PCR 28ccggatcccc cttggtgctg ccctccccgc tgccgggctt cccgctcccg ctggtgctgc 60ccaggaacag gtggtggcgg ccctcg 86 29 24 DNA Artificial Sequence Primer forPCR 29 gtacgacgat gacgataagg atcc 24

What is claimed is:
 1. A non-oligomerizing tandem fluorescent protein,comprising a first monomer of a fluorescent protein operatively linkedto at least a second monomer of the fluorescent protein, wherein thepropensity of the tandem fluorescent protein to oligomerize is reducedor inhibited as compared to a monomer of the fluorescent protein.
 2. Thenon-oligomerizing tandem fluorescent protein of claim 1, wherein thefluorescent protein is a green fluorescent protein (GFP), a redfluorescent protein (RFP), or a fluorescent protein related to a GFP oran RFP.
 3. The non-oligomerizing tandem fluorescent protein of claim 2,wherein the fluorescent protein is a Discosoma RFP or a fluorescentprotein related to a Discosoma RFP.
 4. The non-oligomerizing tandemfluorescent protein of claim 3, wherein the Discosoma RFP is DsRed,which comprises an amino acid sequence as set forth in SEQ ID NO: 12 ora mutant of SEQ ID NO:12.
 5. The non-oligomerizing tandem fluorescentprotein of claim 3, wherein the Discosoma RFP is a mutant of DsRed,which comprises an amino acid sequence as set forth in SEQ ID NO: 12,further comprising an R mutation.
 6. The non-oligomerizing tandemfluorescent protein of claim 2, wherein the fluorescent protein is anAequorea GFP, a Renilla GFP, a Phialidium GFP, or a fluorescent proteinrelated to an Aequorea GFP, a Renilla GFP, and a Phialidium GFP.
 7. Thenon-oligomerizing tandem fluorescent protein of claim 6, wherein thefluorescent protein related to the Aequorea GFP is a cyan fluorescentprotein (CFP), or a yellow fluorescent protein (YFP), or a spectralvariant of the CFP or the YFP.
 8. The non-oligomerizing tandemfluorescent protein of claim 6, wherein the fluorescent protein relatedto the Aequorea GFP is an enhanced GFP (EGFP; SEQ ID NO: 4), an enhancedCFP (ECFP; SEQ ID NO: 6), an EYFP-V68L/Q69K (SEQ ID NO: 10), or anenhanced YFP (EYFP; SEQ ID NO: 8).
 9. The non-oligomerizing tandemfluorescent protein of claim 1, wherein the fluorescent protein furthercomprises a mutation of an amino acid residue corresponding to A206,L221, F223, or a combination thereof of SEQ ID NO:
 2. 10. Thenon-oligomerizing tandem fluorescent protein of claim 10, wherein themutation corresponds to an A206K mutation, an L221K mutation, an F223Rmutation, or an L221K and F223R mutation of SEQ ID NO:
 2. 11. Thenon-oligomerizing tandem fluorescent protein of claim 10, wherein themutation corresponds to an A206K mutation, an L221K mutation, an F223Rmutation, or an L221K and F223R mutation of SEQ ID NO: 6 or SEQ ID NO:10.
 12. The non-oligomerizing tandem fluorescent protein of claim 1,wherein the first monomer and the second monomer are operatively linkedusing a peptide linker.
 13. The non-oligomerizing tandem fluorescentprotein of claim 12, wherein the fluorescent protein is DsRed, whichcomprises an amino acid sequence as set forth in SEQ ID NO:12.
 14. Thenon-oligomerizing tandem fluorescent protein of claim 13, wherein thepeptide linker has an amino acid sequence as set forth in SEQ ID NO:26.15. The non-oligomerizing tandem fluorescent protein of claim 1, furthercomprising at least a third monomer of the fluorescent protein, which isoperatively linked to the first monomer or the second monomer.
 16. Afusion protein, comprising the non-oligomerizing tandem fluorescentprotein of claim 1 operatively linked to at least one polypeptide ofinterest.
 17. The fusion protein of claim 16, wherein thenon-oligomerizing tandem fluorescent protein is linked to thepolypeptide of interest through a peptide bond.
 18. The fusion proteinof claim 17, wherein the non-oligomerizing tandem fluorescent protein islinked to the polypeptide of interest through a linker molecule.
 19. Thefusion protein of claim 16, wherein the at least one polypeptide ofinterest comprises a peptide tag.
 20. The fusion protein of claim 19,wherein the peptide tag is a polyhistidine peptide.
 21. The fusionprotein of claim 16, wherein the polypeptide of interest is a cellularpolypeptide.
 22. The fusion protein of claim 16, wherein the polypeptideof interest is an enzyme, a G-protein, a growth factor receptor, or atranscription factor.
 23. The fusion protein of claim 16, wherein thepolypeptide of interest is one of two or more proteins that associate toform a complex.
 24. A kit, comprising at least one non-oligomerizingtandem fluorescent protein of claim
 1. 25. The kit of claim 24,comprising a plurality of different non-oligomerizing tandem fluorescentproteins.
 26. The kit of claim 24, wherein the non-oligomerizing tandemfluorescent protein comprises a fusion protein.
 27. A polynucleotideencoding the non-oligomerizing tandem fluorescent protein of claim 1.28. A polynucleotide encoding the non-oligomerizing tandem fluorescentprotein of claim
 4. 29. A vector, comprising the polynucleotide of claim27.
 30. A host cell containing the polynucleotide of claim
 27. 31. Akit, comprising at least one polynucleotide of claim
 27. 32. Arecombinant nucleic acid molecule, comprising the polynucleotide ofclaim 27 operatively linked to at least a second polynucleotide.
 33. Therecombinant nucleic acid molecule of claim 32, wherein the at leastsecond polynucleotide comprises a transcription or translationregulatory element.
 34. The recombinant nucleic acid molecule of claim32, wherein the at least second polynucleotide encodes a polypeptide ofinterest.
 35. A vector, comprising the recombinant nucleic acid moleculeof claim
 32. 36. The vector of claim 35, wherein the vector is anexpression vector.
 37. A host cell containing the recombinant nucleicacid molecule of claim
 32. 38. A kit, comprising at least onerecombinant nucleic acid molecule of claim
 32. 39. The kit of claim 38,wherein the at least second polynucleotide comprises a restrictionendonuclease recognition site or a recombinase recognition site.
 40. Thekit of claim 38, wherein the at least second polynucleotide encodes apolypeptide of interest.
 41. The kit of claim 40, wherein the at leastsecond polynucleotide encodes a peptide tag.
 42. The kit of claim 38,comprising a plurality of different recombinant nucleic acid molecules.43. A tandem non-oligomerizing fluorescent protein, comprising: a donor,comprising a first fluorescent protein, an acceptor, comprising a secondfluorescent protein, and a peptide linker moiety operatively linking thedonor and the acceptor, wherein the first fluorescent protein and secondfluorescent protein are different, wherein at least the firstfluorescent protein or the second fluorescent protein is anon-oligomerizing tandem fluorescent protein of claim 1, whereincyclized amino acids of the donor emit light characteristic of thedonor, and wherein the donor and the acceptor exhibit fluorescenceresonance energy transfer when the donor is excited, and the linkermoiety does not substantially emit light to excite the acceptor.
 44. Thetandem non-oligomerizing fluorescent protein of claim 43, wherein eachof the first fluorescent protein and the second fluorescent protein is anon-oligomerizing tandem fluorescent protein.
 45. The tandemnon-oligomerizing fluorescent protein of claim 43, wherein thenon-oligomerizing tandem fluorescent protein comprises a Discosoma RFPor a fluorescent protein related to a Discosoma RFP.
 46. The tandemnon-oligomerizing fluorescent protein of claim 45, wherein the DiscosomaRFP is DsRed, which comprises an amino acid sequence as set forth in SEQID NO: 12 or a mutant of SEQ ID NO:12.
 47. The non-oligomerizing tandemfluorescent protein of claim 45, wherein the Discosoma RFP is a mutantof DsRed, which comprises an amino acid sequence as set forth in SEQ IDNO: 12, further comprising an I125R mutation.
 48. The tandemnon-oligomerizing fluorescent protein of claim 43, wherein the firstfluorescent protein is a non-oligomerizing tandem fluorescent protein,and the second fluorescent protein is a non-oligomerizing fluorescentprotein.
 49. The tandem non-oligomerizing fluorescent protein of claim48, wherein the non-oligomerizing fluorescent protein comprises amutation of an amino acid residue corresponding to A206, L221, F223, ora combination thereof of SEQ ID NO:2.
 50. The tandem non-oligomerizingfluorescent protein of claim 49, wherein the mutation corresponds toS65G/S72A/T203Y/H231L in SEQ ID NO:2.
 51. The tandem non-oligomerizingfluorescent protein of claim 49, wherein the mutation corresponds toS65G/V68L/Q69K/S72A/T203Y/H231L in SEQ ID NO:2.
 52. The tandemnon-oligomerizing fluorescent protein of claim 49, wherein the mutationcorresponds to K26R/F64L/S65T/Y66W/N146I/M153T/V163A/N164H/H231L in SEQID NO:
 2. 53. The tandem non-oligomerizing fluorescent protein of claim49, wherein the mutation corresponds to H148G in SEQ ID NO:
 2. 54. Amethod for determining the pH of a sample, the method comprising:contacting the sample with a first non-oligomerizing tandem fluorescentprotein of claim 1, wherein the emission intensity of the firstnon-oligomerizing tandem fluorescent protein changes as pH variesbetween pH 5 and pH 10, exciting the indicator; and determining theintensity of light emitted by the first non-oligomerizing tandemfluorescent protein at a first wavelength, wherein the emissionintensity of the first non-oligomerizing tandem fluorescent proteinindicates the pH of the sample.
 55. The method of claim 54, wherein thefirst non-oligomerizing tandem fluorescent protein comprises a DiscosomaRFP or a fluorescent protein related to a Discosoma RFP.
 56. The methodof claim 55, wherein the Discosoma RFP is DsRed, which comprises anamino acid sequence as set forth in SEQ ID NO: 12 or a mutant of SEQ IDNO:12.
 57. The method of claim 56, wherein the mutant of SEQ ID NO: 12comprises an I125R mutation.
 58. The method of claim 54, wherein thesample is a biological tissue.
 59. The method of claim 54, wherein thesample is a cell or a fraction thereof.
 60. The method of claim 54,further comprising: contacting the sample with a non-oligomerizingfluorescent protein, wherein the non-oligomerizing fluorescent proteinis different from the first non-oligomerizing tandem fluorescentprotein, wherein the emission intensity of the non-oligomerizingfluorescent protein changes as pH varies from 5 to 10, and wherein thenon-oligomerizing fluorescent protein emits at a second wavelength thatis distinct from the first wavelength; exciting the non-oligomerizingfluorescent protein; determining the intensity of light emitted by thenon-oligomerizing fluorescent protein at the second wavelength; andcomparing the fluorescence at the second wavelength to the fluorescenceat the first wavelength.
 61. The method of claim 60, wherein thenon-oligomerizing fluorescent protein is a second non-oligomerizingtandem fluorescent protein.
 62. The method of claim 54, wherein thefirst non-oligomerizing tandem fluorescent protein comprises a targetingsequence.
 63. The method of claim 62, wherein the targeting sequencecomprises a cell compartmentalization domain.
 64. The method of claim63, wherein the cell compartmentalization domain targets the firstnon-oligomerizing tandem fluorescent protein in a cell to cytosol,endoplasmic reticulum, mitochondrial matrix, chloroplast lumen, medialtrans-Golgi cistemae, a lumen of a lysosome, or a lumen of an endosome.65. The method of claim 64, wherein the cell compartmentalization domaincomprises amino acid residues 1 to 81 of human type II membrane-anchoredprotein galactosyltransferase, or amino acids 1 to 12 of the presequenceof subunit IV of cytochrome c oxidase.
 66. A method for determiningwhether a sample contains an enzyme, the method comprising: contacting asample with a tandem non-oligomerizing fluorescent protein of claim 43;exciting the donor, and determining a fluorescence property in thesample, wherein the presence of the enzyme in the sample results in achange in the degree of fluorescence resonance energy transfer.
 67. Amethod for determining the activity of an enzyme in a cell, the methodcomprising: providing a cell that expresses a tandem non-oligomerizingtandem fluorescent protein of claim 43, wherein the peptide linkermoiety comprises a cleavage recognition amino acid sequence specific forthe enzyme coupling the donor and the acceptor, exciting the donor, anddetermining the degree of fluorescence resonance energy transfer in thecell, wherein the presence of enzyme activity in the cell results in achange in the degree of fluorescence resonance energy transfer.
 68. Amethod for identifying the presence of a molecule in a sample, themethod comprising: operatively linking a non-oligomerizing tandemfluorescent protein of claim 1 to the molecule, and detectingfluorescence due to the non-oligomerizing tandem fluorescent protein ina sample suspected of containing the molecule, thereby identifying thepresence of the molecule in the sample.
 69. The method of claim 68,wherein the molecule is a polypeptide.
 70. The method of claim 69,wherein the polypeptide is an antibody, an enzyme, or a receptor. 71.The method of claim 68, wherein the molecule is a polynucleotide. 72.The method of claim 68, wherein the sample is a biological sample. 73.The method of claim 72, wherein the biological sample comprises a cell,a tissue sample, or an extract of a cell or a tissue sample.
 74. Themethod of claim 73, wherein said detecting is performed on an intactcell or tissue sample.
 75. The method of claim 68, wherein saidoperatively linking comprises contacting the non-oligomerizing tandemfluorescent protein with the molecule under conditions suitable forlinking the protein to the molecule.
 76. The method of claim 68, whereinsaid operatively linking comprises expressing a recombinant nucleic acidmolecule comprising a polynucleotide encoding the non-oligomerizingtandem fluorescent protein operatively linked to a polynucleotideencoding the molecule.
 77. A method of identifying an agent or conditionthat regulates the activity of an expression control sequence, themethod comprising: exposing a recombinant nucleic acid moleculecomprising a polynucleotide encoding a non-oligomerizing tandemfluorescent protein of claim 1 operatively linked to an expressioncontrol sequence to an agent or condition suspected of being able toregulate expression of a polynucleotide from the expression controlsequence, and detecting fluorescence of the non-oligomerizing tandemfluorescent protein due to said exposing, thereby identifying an agentor conditions that regulates expression of the expression controlsequence.
 78. The method of claim 77, wherein the expression controlsequence is a transcription regulatory element.
 79. The method of claim78, wherein the transcription regulatory element is a promoter.
 80. Themethod of claim 77, wherein the expression control sequence is atranslation regulatory element.
 81. The method of claim 77, wherein thecondition comprises exposure to proteins expressed in a cell.
 82. Amethod of identifying a specific interaction of a first molecule and asecond molecule, the method comprising: contacting the first molecule,which is operatively linked to a donor first non-oligomerizing tandemfluorescent protein, and the second molecule, which is operativelylinked to an acceptor non-oligomerizing fluorescent protein, underconditions that allow a specific interaction of the first molecule andsecond molecule, wherein the first non-oligomerizing tandem fluorescentprotein and the non-oligomerizing fluorescent protein are different;exciting the donor; and detecting fluorescence resonance energy transferfrom the donor to the acceptor, thereby identifying a specificinteraction of the first molecule and the second molecule.
 83. Themethod of claim 82, wherein the non-oligomerizing fluorescent protein isa second non-oligomerizing tandem fluorescent protein.
 84. The method ofclaim 82, wherein the first molecule is a first cellular protein and thesecond molecule is a second cellular protein.
 85. The method of claim82, wherein first cellular protein and the second cellular protein arethe same.
 86. The method of claim 82, wherein the first molecule is apolynucleotide and the second molecule is a polypeptide.
 87. The methodof claim 86, wherein the polynucleotide is a transcription regulatoryelement and the polypeptide is a putative transcription factor.